Arrdc1-mediated microvesicles (armms) and uses thereof

ABSTRACT

The invention provide isolated arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMMs). Methods for generating and for isolating ARMMs are also provided herein. ARMMs can be used to deliver agents, for example, nucleic acids (e.g., siRNAs, microRNAs, lincRNAs), proteins (e.g., transcription factors, chromatin modulators, kinases, phosphorylases, or recombinases), or small molecules to target cells in vitro and in vivo, and methods for such ARMM-mediated delivery are provided herein. Diagnostic and therapeutic methods using ARMMs are also described herein.

RELATED APPLICATION

This application is a continuation of and claims priority under 35U.S.C. § 120 to U.S. patent application Ser. No. 15/669,720, filed Aug.4, 2017, which is a divisional of and claims priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 14/376,967, filed Aug. 6, 2014,now U.S. Pat. No. 9,737,480, which is a national stage filing under 35U.S.C. § 371 of international PCT application, PCT/US2013/024839, filedFeb. 6, 2013, which claims priority under 35 U.S.C. § 119(e) to U.S.provisional patent application Ser. No. 61/595,416, filed Feb. 6, 2012,each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under ES016454 awardedby the National Institutes of Health and HDTRA1-06-C-0039 awarded by theDepartment of Defense/Defense Threat Reduction Agency. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Mammalian cells are capable of delivering multiple types of membranecapsules, also referred to as microvesicles, extracellularly. Suchmicrovesicles shed by one cell can be taken up by another cell, thusallowing for intercellular communication and transport of moleculescontained within the microvesicles. For example, the limiting membraneof late endosomes can fuse with the plasma membrane, leading to theextracellular release of multivesicular bodies (MVBs), initiallycontained within the endosomes, as exosomes. Also, budding virusesexploit the tumor susceptibility gene 101 (TSG101) protein and thehighly conserved ESCRT (Endosomal Sorting Complex Required forTransport) machinery used for MVB formation to mediate the egress ofviral particles from host cells.

SUMMARY OF THE INVENTION

Some aspects of this invention relate to the discovery of avirus-independent cellular process that generates microvesicles that aredistinct from exosomes and which, like budding viruses, are produced bydirect plasma membrane budding (DPMB). DPMB is driven by a specificinteraction of TSG101 with a tetrapeptide PSAP motif of an accessoryprotein, the arrestin-domain-containing protein ARRDC1—which, asdescribed herein, is localized to the plasma membrane through itsarrestin domain. The ARRDCII./TSG101 interaction results in relocationof TSG101 from endosomes to the plasma membrane and mediates the releaseof microvesicles that contain TSG101, ARRDC1, and other cellularcomponents, including, for example, proteins and nucleic acids.

Unlike exosomes, which are derived from MVBs, ARRDC1-mediatedmicrovesicles (ARMMs) lack known late endosomal markers. ARUM formationinvolves VPS4 ATPase and is enhanced by the E3 ligase WWP2, whichinteracts with and ubiquitinates ARRDC1. Some aspects of this inventionrelate to the discovery of an intrinsic cellular mechanism that resultsin direct budding of microvesicles from the plasma membrane, providing aformal paradigm for the evolutionary recruitment of ESCRT proteins inthe release of budding viruses. Some aspects of this invention relate tothe surprising discovery that ARRDC1 protein discharged into ARMMs canbe transferred to co-cultured cells, suggesting a role for ARMMs inintercellular communication, and allowing for the use of ARMMs asvectors for the delivery of various biomolecules (e.g., proteins,polypeptides, nucleic acids, RNAi agents) to a target cell without theneed for genetically modifying a target cell. Other agents such as, forexample, small molecules, can also be delivered via ARMMs,

In some aspects of this invention isolated ARMMs are provided. SuchARMMs may be isolated from a subject, a biological sample, or a cellculture, or ARMMs may be prepared synthetically. Methods for generatingand/or isolating ARMMs, including ARMMs that include an agent to bedelivered to a target cell or target cell population, are also providedherein. Methods for the use of ARMMs to deliver agents, for example,nucleic acids (e.g, siRNAs, microRNAs, lincRNAs), proteins or peptides(e.g., transcription factors, chromatin modulators, kinases,phosphorylases, or recombinases), or small molecules to target cells invitro and in vivo are also provided, as are diagnostic and therapeuticmethods using ARMMs. Some aspects of this invention provide an isolatedarrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle(ARMM) comprising a lipid bilayer and an ARRDC1 protein, or fragmentthereof. In some embodiments, the ARRDC1 protein or fragment thereofcomprises an ARRDC1 PSAP domain. In some embodiments, the ARMM furthercomprises a TSG101 protein or fragment thereof. In some embodiments, theTSG101 protein fragment comprises a TSG101 UEV domain. In someembodiments, the ARMM further comprises a cell surface protein (e.g.,receptor) or a cytosolic protein (e.g., enzyme). In some embodiments,the ARMM further comprises an integrin, a receptor tyrosine kinase, aG-protein coupled receptor, a membrane-bound immunoglobulin, or aprotein listed in Table 1, In some embodiments, the microvesiclecomprises an integrin chosen from the group consisting of (α1β1, α2β1,α4β1, α5β1, α6β1, αLβ2, αMβ2, αIIbβ3, αVβ3, αVβ5, αVβ6, and α6β4integrins; a receptor tyrosine kinase chosen from the group consistingof an EGF receptor (ErbB family), insulin receptor, .PDGF receptor, FGFreceptor, VEGF receptor, HGF receptor, Trk receptor, Eph receptor, AXL,receptor, LTK receptor, TIE receptor, ROR receptor, DDR receptor, RETreceptor, KLG receptor, RYK receptor, and MuSK receptor; a G-proteincoupled receptor chosen from the group consisting of a rhodopsin-likereceptor, secretin receptor, metabotropic glutamate/pheromone receptor,cyclic AMP receptor, frizzled/smoothened receptor, CXCR4, CCR5, orbeta-adrenergic receptor; and/or an exocyst protein listed in Table 1chosen from EXOC7, EXOC8, EXOC1, and EXOC2.

In some embodiments, the ARMM does not include an exosomal biomarker. Insome embodiments, the ARMM does not include one or more exosomalbiomarkers. In some embodiments, the exosomal biomarker is chosen fromthe group consisting of CD63, Lamp-1, Lamp-2, CD9, HSPA8, GAPDH, CD81,SDCBP, PDCD6IP, ENO1, ANXA2, ACTB, YWHAZ, HSP90AA1, ANXA5, EEF1A1,YWHAE, PPIA., MSN, CFL1, ALDOA, PGK1, EEF2, ANXA1, PKM2, HLA-DRA, andYWHAB.

Some embodiments of this invention provide ARMMs comprising an agent,for example, a protein, a nucleic acid, or a small molecule. In someembodiments, the agent is conjugated to the ARRDC1protein, the ARRDC1fragment, the TSG101 protein, or the TSG101 fragment. In someembodiments, the nucleic acid is an RNA. In some embodiments, thenucleic acid is an RNAi agent. In some embodiments, the nucleic acid isa coding RNA, a non-coding RNA, an antisense RNA, an mRNA, a small RNA,an siRNA, an shRNA, a microRNA, an snRNA, a snoRNA, a lincRNA, astructural RNA, a ribozyme, or a precursor thereof. In some embodiments,the nucleic acid is a DNA. In some embodiments, the nucleic acidcomprises a restrotransposon sequence, a LINE sequence, a SINE sequence,a composite SINE sequence, or an LTR-retrotransposon sequence. In someembodiments, the nucleic acid encodes a protein. In some embodiments,the agent comprises a detectable label. In some embodiments, the agentis a therapeutic agent. In some embodiments, the agent is a drugapproved for human or veterinary use by a governmental agency. In sonicembodiments, the agent is a cytotoxic agent. In some embodiments, theagent is a protein. In some embodiments, the agent is a transcriptionfactor, a transcriptional repressor, a fluorescent protein, a kinase, aphosphatase, a protease, a ligase, or a recombinase. In someembodiments, the agent is a small molecule. In some embodiments, theagent is covalently bound to the ARRDC1 protein or fragment thereof, theTSG101 protein or fragment thereof, or another protein of the ARMM. Insome embodiments, agent is conjugated to the ARRDC1 protein or fragmentthereof, the TSG101 protein or fragment thereof, or other protein via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the linker comprises a protease recognition site. In someembodiments, the linker is a UV-cleavable linker. In certainembodiments, the linker is cleaved under specific conditions such as pH,redox conditions, etc. In some embodiments, the microvesicle diameter isfrom about 30 nm to about 500 nm.

Some aspects of this invention provide an ARRDC1 fusion protein thatcomprises an ARRDC1 protein or a fragment thereof, and a polypeptideconjugated to the ARRDC1 protein or fragment thereof. In someembodiments, the ARRDC1 fragment comprises a PSAP domain. Some aspectsof this invention provide a TSG101 fusion protein, comprising a TSG101protein or a fragment thereof, and a polypeptide conjugated to theTSG101 protein or fragment thereof. In some embodiments, the TSG101fragment comprises a UEV domain. In some embodiments, the conjugatedpolypeptide comprises a transcription factor, a transcriptionalrepressor, a fluorescent protein, a kinase, a phosphatase, a protease, aligase, or a recombinase. In some embodiments, the polypeptide isconjugated to the ARRDC1 protein, the ARRDC1 protein fragment, theTSG101 protein, the TSG101 protein fragment, or other ARMM-associatedprotein via a covalent bond. In some embodiments, the polypeptide isconjugated to the ARRDC1 protein, the ARRDC1 protein fragment, theTSG101 protein, or the TSG101 protein fragment via a linker. In someembodiments, the linker is a cleavable linker.

Some aspects of this invention provide a microvesicle-producing cellthat comprises a recombinant expression construct encoding an ARRDC1protein, or a PSAP domain-comprising fragment thereof, under the controlof a heterologous promoter. In some embodiments, the expression ofARRDC1 induces or increases ARMM production of the cell. In someembodiments, the cell expresses or contains an agent in its cytoplasm orits plasma membrane that is included in ARMMs produced by the cell. Someaspects of this invention provide a microvesicle-producing cell thatcomprises a recombinant expression construct encoding a TSG101 protein,or a UEV domain-comprising fragment thereof, under the control of aheterologous promoter. In some embodiments, the expression constructfurther encodes a polypeptide fused to the ARRDC1 protein or the TSG101protein. In some embodiments, the polypeptide comprises a transcriptionfactor, a transcriptional repressor, a fluorescent protein, a kinase, aphosphatase, a protease, a ligase, or a recombinase. In someembodiments, the cells is a eukaryotic cell. In some embodiments, thecell is a mammalian cell. In some embodiments, the cell is a human cell.In some embodiments, the cell further comprises a recombinant expressionconstruct encoding an RNAi agent. In some embodiments, the RNAi agent isa nucleic acid. In some embodiments, the nucleic acid is a non-codingRNA, an antisense RNA, a small RNA, an siRNA, an shRNA, a microRNA, ansnRNA, a snoRNA, a lincRNA, or a precursor thereof. In some embodiments,the cell further comprises a recombinant expression construct encoding aribozyme.

Some aspects of this invention provide methods of delivering an agent toa target cell comprising contacting the target cell with a microvesiclewith an agent to be delivered, as described herein. In other aspects ofthis invention provided are methods of delivering an agent to a targetcell by exposing the target cell to a microvesicle-producing cell (e.g,by co-culturing both cells). In certain embodiments, the inventionprovides methods of delivering an agent to a target cell comprisingcontacting the target cell with an isolated microvesicle that comprisesa lipid bilayer, an ARRDC1 protein or fragment thereof, and the agent tobe delivered. In certain embodiments, the target cell is a mammaliancell. In some embodiments, the target cell is a human cell. In someembodiments, the target cell is a stem cell. In some embodiments, thetarget cell is a cell in vitro or ex vivo, and the method comprisesadministering the microvesicle to the cell in vitro, or co-culturing thetarget cell with the microvesicle-producing cell in vitro. In someembodiments, the target cell is a cell in a subject, and the methodcomprises administering the microvesicle or the microvesicle-producingcell to the subject. In some embodiments, the subject is a mammaliansubject. In some embodiments, the subject is a human subject. In someembodiments, the target cell is a pathological cell. In someembodiments, the target cell is a cancer cell. In some embodiments, themicrovesicle includes a targeting agent that selectively binds anantigen of the target cell. In some embodiments, the antigen of thetarget cell is a cell surface antigen. In some embodiments, thetargeting agent is a membrane-bound immunoglobulin, an integrin, areceptor, a receptor ligand, an aptamer, a small molecule, or a fragmentthereof.

Some aspects of this invention provide an in vitro cell culture systemcomprising (a) a microvesicle-producing cell population comprising arecombinant expression construct encoding (i) an ARRDC1 protein orfragment thereof under the control of a heterologous promoter, and/or(ii) a TSG101 protein or fragment thereof under the control of aheterologous promoter; and (b) a target cell population. In someembodiments, the ARRDC1 fragment comprises a PSAP domain, and/or theTSG101 fragment comprises a UEV domain. In some embodiments, theexpression construct further encodes a polypeptide fused to the ARRDC1protein or fragment thereof. In some embodiments, the expressionconstruct further encodes a polypeptide fused to the TSG101 protein orfragment thereof. In some embodiments, the polypeptide fused to theARRDC1 protein or fragment, or to the TSG101 protein or fragment,independently comprises a transcription factor, a transcriptionalrepressor, a fluorescent protein, a kinase, a phosphatase, a protease, aligase, or a recombinase. In some embodiments, themicrovesicle-producing cell is a mammalian cell. In some embodiments,the microvesicle-producing cell is a non-proliferating cell. In someembodiments, the microvesicle-producing cell is a feeder cell. In someembodiments, the microvesicle-producing cell is an immortalized cell.For example, in some embodiments, the microvesicle-producing cell is aHEK293T cell, a HeLa cell, or an A549 cell. In some embodiments, thetarget cell is a mammalian cell. In some embodiments, the target cell isa human cell. In some embodiments, the target cell is a differentiatedcell. In some embodiments, the target cell is a stem cell.

In yet other aspects, the invention provides methods of detecting acondition in a subject. The detection of ARMMs or certaincharacteristics of the ARMM may be used to diagnose a specific conditionor disease in a subject. For example, the detection of ARMMs may be usedto diagnose cancer in subject. In certain embodiments, the inventivemethod comprises (a) obtaining or detecting an ARMM from the subject ora biological sample from the subject; and (b) detecting a biomarkerprofile indicative of the condition in the ARMM obtained from thesubject; wherein the presence of the biomarker profile in the ARMMobtained from the subject indicates the presence of the condition in thesubject. In some embodiments, the method further comprises obtaining abiological sample from the subject that comprises an ARMM. In someembodiments, a method is provided that comprises detecting the presenceof an ARMM or a level of ARMMs to diagnose a disease. Some aspects ofthis invention provide methods of detecting a pathological cell or cellpopulation in a subject comprising (a) detecting a level of ARMMsproduced by a cell or cell population obtained from the subject; and (b)comparing the level of ARMMs to a control level, wherein, if the levelof ARMMs produced by the cell or cell population obtained from thesubject is higher than the control level, then the cell or cellpopulation is indicative of a pathological cell or cell population. Insome embodiments, the method further comprises obtaining a sample fromthe subject comprising a cell or cell population producing ARMMs. Someaspects of this invention provide methods of detecting a pathologicalcell or cell population in a subject comprising (a) obtaining an ARMMfrom the subject; and (b) detecting a biomarker profile indicative ofthe pathological cell or cell population in the ARMM obtained from thesubject; wherein the presence of the biomarker profile in the ARMMobtained from the subject indicates the presence of the pathologicalcell or cell population condition in the subject. In some embodiments,the method further comprises obtaining a sample from the subjectcomprising an ARMM. In some embodiments, the pathological cell or cellpopulation is a malignant cell or cell population.

Some aspects of this invention provide an expression constructcomprising (a) a nucleotide sequence encoding an ARRDC1 protein, orfragment thereof operably, linked to a heterologous promoter, and (b) arestriction site or a recombination site positioned adjacent to theARRDC1-encoding nucleotide sequence allowing for the insertion of anucleotide sequence encoding an additional polypeptide in frame with theARRDC1-encoding nucleotide sequence. Such expression constructs areuseful for generating ARRDC1 fusion proteins that can be expressed in acell, which, in turn, induces or increases ARMM production in the cell.An ARMM produced by a cell expressing such an ARRDC1 fusion proteinwill, in some embodiments, comprise the ARRDC1 fusion protein encoded bythe expression construct. ARRDC1 and TSG101 fusion proteins can be usedas research tools to investigate ARMM generation or trace the respectivefusion proteins or the ARMMs that they are incorporated into. Forexample, in some embodiments, an expression construct encoding an ARRDC1or TSG101. fusion protein comprising a fluorescent protein are provided.Some aspects of this invention provide an expression constructcomprising (a) a nucleotide sequence encoding a TSG101 protein orfragment thereof operably linked to a heterologous promoter, and (b) arestriction site or a recombination site positioned adjacent to theTSG101 -encoding nucleotide sequence allowing for the insertion of anucleotide sequence encoding an additional polypeptide in frame with theTSG101-encoding nucleotide sequence.

Other advantages, features, and uses of the invention will be apparentfrom the detailed description of certain exemplary, non-limitingembodiments; the drawings; the non-limiting working examples; and theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. ARRDC1interacts with and recruits TSG101 to the cellmembrane via a conserved PSAP motif. (FIG. 1A) Schematic representationof the ARRDC1 protein, indicating relevant structural domains: theN-terminal arrestin domain (gray box), and the C-terminal PSAP motif(filled box), and PPXY motifs (open boxes). Also depicted are the ARRDC1fragments identified in the yeast two-hybrid screen using the UEV domainof TSG101 as the bait. The sequences, from left to right, correspond toSEQ ID NOs: 37-39. (FIG. 1B) Alignment of fragments of ARRDC1 orthologsshowing conservation of the PSAP motif. Sequences correspond, from topto bottom, to SEQ NOs: 8 to 13 (FIG. 1C) Western blot analysis of theinteraction between FLAG-tagged wild-type (wt) ARRDC1 or the PSAP mutant(PAAP (SEQ ID NO: 32)) and HA-tagged TSG-101. Anti-FLAGimmunoprecipitates and whole cell extracts (WCE) from 293T cellstransfected with the indicated constructs were examined byimmunoblotting using the indicated antibodies. (FIG. 1D) Western blotanalysis of the interaction between HA-tagged wild type or a mutant(M95A) TSG-101 with ARRDC1, as in (FIG. 1C). (FIG. 1E) Localization ofmCherry-TSG101 fusion protein in 293T cells co-transfected with GFP,ARRDC1-GFP, or its PSAP mutant (PAAP (SEQ ID NO: 32)). Cells were fixedand examined by confocal microscopy. More than twenty cells expressingboth ARRDC1-GFP and mcherry-TSG101 were examined and all showed similarlocalization pattern. Representative images are shown in the figure.

FIGS. 2A-2E. ARRDC1expression potentiates release of vesicles that aredevoid of exosomal markers and can fuse with recipient cells. (FIG. 2A)Western blot analysis, using the indicated antibodies, of whole cellextracts and of the corresponding extracellular vesicles produced by293T cells transfected with constructs expressing GFP, wt ARRDC1 -GEP(wt), or HIV GAG-GFP. (FIG. 2B) Electron microscopy of extracellularvesicles derived from 293T cells transfected with an ARRDC1 expressionconstruct. Scale bar=100 nm. (FIG. 2C) Western blot analysis of wholecell extracts (WCEs) and extracellular vesicles from 293T cellstransfected with non-targeting shRNA. or ARRDC 1-targeting shRNAlentiviruses. (FIG. 2D) Anti-ARRDC1 and anti-CD63 immuogold labeling ofcollagen-embedded extracellular vesicle cryosections. Arrows indicateimmunogold-positive labeling, Scale bar=100 nm. (FIG. 2E) Transfer ofARRDC1-containing vesicles from donor to recipient cells. Cellstransfected by increasing amounts of constructs expressing ARRDC1-GFP orGFP were washed and seeded in 0.4 μm transwells atop untransfectedcells. Western blot analysis of the corresponding lysates fromtransfected donor cells and untransfected recipient cells was carriedout using the indicated antibodies.

FIGS. 3A-3C. The ARRDC1-TSG101 interaction and the budding-relatedprotein VPS4a are required for release of ARRDC1-regulated microvesicles(ARMMs). (FIG. 3A) ARRDC1 and TSG101 are required for ARMMs release.Western blot analysis of ARMMs and whole cell extracts (WCE) from 293Tcells subjected to transfection by indicated siRNAs. The results shownare representative of three independent replication experiments. (FIG.3B) PSAP mutant ARRDC1 is released less efficiently into ARMMs. Cellswere transfected with wt or PSAP mutant (PAAP (SEQ ID NO: 32))ARRDC1-GFP and the corresponding WCEs and ARMMs were analyzed by Westernblotting probed by the indicated antibodies. (FIG. 3C) ARMMs releaserequires functional VPS4 ATPase activity. 293T cells were co-transfectedwith constructs expressing HA-ARRDC1 and 0.5 or 1 μg of VPS4a or VPS4aE228Q (VPS4a DN) GFP fusion proteins. WCEs and ARMMs were analyzed byWestern blotting using the indicated antibodies.

FIGS. 4A-4C. Arrestin domain -mediated association of ARRDC1with theplasma membrane is essential for cell surface-derived budding of ARMMs.(FIG. 4A) ARMMs bud at the cell surface. Cryosections of 293T cellstransfected with constructs expressing mCherry or ARRDC1-mCherry wereimmunogold labeled with anti-mCherry. White arrows in the right panelindicate presumptive outward budding at the cell membrane adjacent toimmunogold-positive ARMMs captured from the intercellular space. Anuntransfected cell is indicated in the same field and shows no staining.Scale bar=100 nm. (FIG. 4B) Disruption of conserved arrestin residuesalters localization of ARRDC1. Conserved arrestin domain residues amongarrestins 1-4 and the known membrane-associated ARRDC1 and. ARRDC3 wereidentified by ClustalW alignment and were mutated. The correspondingmCherry fusion mutants were expressed in 293T cells and showed alteredlocalization compared to wt or PAAP ARRDC1. The sequences in theschematic, from top to bottom and left to right, correspond to SEQ IDNOs: 14-31. (FIG. 4C) 293T cells were transfected with constructsexpressing the indicated ARRDC1mutants fused to mCherry. Thecorresponding ARMMs and WCEs were analyzed by Western blotting with theindicated antibodies. The sequence corresponds to SEQ ID NO: 32.

FIGS. 5A-5B. WWP2 interacts with and ubiquitinates ARRDC1 to enhancesARMMs release. (FIG. 5A) Deletion of PPXY motifs reduced ARRDC1ubiquitination and ARMMs release, 293T cells were transfected with wt orPPXY deletion (ΔΔPPXY) ARRDC1 fused to mCherry and visualized byconfocal microscopy. The corresponding cell lysates and ARMMs wereanalyzing by Western blotting as indicated. (FIG. 5B) PPXY motifs inARRDC1 interact with WWP2. 293T cells were co-transfected withGFP-tagged WWP2 and HA-tagged wt or ΔΔPPXY ARRDC1. Anti-HAimmunoprecipitates and WCEs were examined by Western blotting asindicated. WWP2 expression potentiates ARRDC1 ubiquitination in ARMMs.293T cells were co-transfected with constructs expressing HA-ARRDC1 andWWP2-GFP or the WW domain of WWP2 fused to GFP. WCEs and ARMMs from eachtransfected group were analyzed by immunoblotting as indicated. WWP2knockdown diminishes ARRDC1 in ARMMs. 293T was subjected to transfectionwith non-targeting (NT) or WWP2-targeting siRNAs. Corresponding WCEs andARMMs were analyzed by Western blotting.

FIG. 6. A model for ARRDC1-mediated ARMMs formation and Gag-mediatedviral budding. Both ARRDC1and Gag interact with TSG101, areubiquitinated by HECT domain ligases, and require VPS4 ATPase to producebudding vesicles. The sequences listed from left to right correspond toSEQ ID NOs: 37 and 40.

FIGS. 7A-7B. Plasma membrane localization of ARRDC1. (FIG. 7A) 293Tcells were seeded on cover slips and transfected with 20 ng ofFlag-ARRDC1 expression plasmid. 48 h later, cells were fixed,permeabilized, and stained with anti-ARRDC1primary antibody (1:200) orno primary as a control, followed by incubation with secondaryanti-Rabbit IgG antibody conjugated to TRITC (Sigma; 1:500). Cover slipswere mounted on glass slides and visualized by confocal microscopy.(FIG. 7B) 293T, HeLa, and A549 cells were seeded on cover slips,transfected with 100 ng of the ARRDC1-GFP construct, and visualized byconfocal microscopy. DAPI staining was used to visualize the nuclei.

FIG. 8. ARRDC1 ubiquitination in ARMMs. 293T cells were co-transfectedwith HA-ubiquitin and GFP or ARRDC1-GFP. Cells and ARMMs were laterlysed in NP-40 lysis buffer. ARMMs lysates were pre-cleared with proteinA agarose beads and subjected to an anti-HA immunoprecipitation tocollect ubiquitinated proteins. Cell lysates, ARMM lysates, and anti-HAARMMs immunoprecipitates were analyzed by Western blotting with theindicated antibodies.

FIG. 9. Self-association of ARRDC1. 293T cells were transfected with thespecified plasmids and then harvested in NP-40 lysis buffer. Lysateswere pre-cleared and then subjected to an anti-FLAG immunoprecipitation(IP). Corresponding samples were analyzed by Western blotting withindicated antibodies. WCE: whole cell extracts.

FIG. 10, Sucrose density gradient separation of ARMMs, ARMMs wereinitially collected through ultracentrifugation of ˜200 ml of mediasupernatant of actively growing HCC1419 cells. ARMMs were then subjectedto further purification by sucrose gradient separation as follows.Briefly, ARMMs were re-suspended in 0.5 ml PBS and laid on top of asucrose step-wise gradient (from 0.2 to 2 M, 1 ml of eachconcentration). The sample was then centrifuged in a swinging bucket(SW50.1 rotor, Beckman) at 130,000×g for 18 hours. After centrifugation,ten (10) fractions of 1 ml volume were collected. A small volume (˜10μl) of each fraction was subjected to SDS-PAGE followed by Westernblotting analysis for ARRDC1.

FIG. 11. SDS-PAGE of purified ARMMs. Following sucrose gradientseparation, two fractions (4, 5, see arrows in FIG. 10) that containmost of ARMMs were combined together, diluted with PBS to a volume of 10ml, and re-centrifuged (100,000×g for 2 hours). The resulting pellet waslysed in SDS-PAGE loading buffer, and proteins were resolved by SDS-PAGE(12% gel) followed by Coomassie-blue staining. About 18 distinct proteinbands were identified and analyzed by mass spectrometry.

FIG. 12. Detection of small RNA species in ARMMs. HEK293 cells werefirst stably transduced with ARRDC1-shRNA to knock down endogenousARRDC1. ARRDC1 -knockdown cells were then transfected with constructsexpressing either mCherry or ARRDC1-mCherry. Supernatant medium fromboth cells were subjected to ultracentrifugation. RNAs in centrifugedpellets were extracted by Trizol reagent (Invitrogen) and analyzed byBioanalyzer (Agilent). Cells expressing ARRDC1-mcherry but not thecontrol mCherry contain detectable RNA species, including small onesranging from 50-80 nucleotides. The cellular RNA profiles from mCherry-and ARRDC1-mCherry-expressing cells are very similar (figure inset).

Definitions

Agent and agent to be delivered: As used herein, the term “agent” refersto a substance that can be incorporated in an ARMM, for example, intothe liquid phase of the ARMM or into the lipid bilayer of the ARMM. Theterm “agent to be delivered” refers to any substance that can bedelivered to a subject, organ, tissue, or cell. In some embodiments, theagent is an agent to be delivered to a target cell. In some embodiments,the agent to be delivered is a biologically active agent, i.e., it hasactivity in a cell, biological system, and/or subject. For instance, asubstance that, when administered to an subject, has a biological effecton that subject, is considered to be biologically active. In someembodiments, an agent to be delivered is a therapeutic agent. As usedherein, the term “therapeutic agent” refers to any agent that, whenadministered to a subject, has a beneficial effect. The term“therapeutic agent” refers to any agent that, when administered to asubject, has a therapeutic, diagnostic, and/or prophylactic effectand/or elicits a desired biological and/or pharmacological effect. Asused herein, the term “therapeutic agent” may be a nucleic acid that isdelivered to a cell via its association with or inclusion into an ARMM.In certain embodiments, the agent to be delivered is a nucleic acid. Incertain embodiments, the agent to be delivered is DNA. In certainembodiments, the agent to be delivered is RNA. In certain embodiments,the agent to be delivered is a peptide or protein. In some embodiments,the functional protein or peptide to be delivered into a cell is atranscription factor, a tumor suppressor, a developmental regulator, agrowth factor, a metastasis suppressor, a pro-apoptotic protein, a zincfinger nuclease, or a recombinase. In some embodiments, the protein tobe delivered is p53, Rb (retinoblastoma protein), BRCA1, BRCA2, PTEN,APC, CD95, ST7, ST14, a BCL-2 family protein, a caspase BRMS1, CRSP3,DRG1, KAI1, KISS1, NM23, a TIMP-family protein, a BMP-family growthfactor, EGF, EPO, FGF, G-CSF, GM-CSF, a GDF-family growth factor, HGF,HDGF, IGF, PDGF, TPO, TGF-α, TGF-β, VEGF; a zinc finger nucleasetargeting a site within the human CCR5 gene, Cre, Dre, or FLPrecombinase. In certain embodiments, the agent to be delivered is asmall molecule. In some embodiments, the agent to be delivered is adiagnostic agent. In some embodiments, the agent to be delivered isuseful as an imaging agent. In some of these embodiments, the diagnosticor imaging agent is, and in others it is not, biologically active.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans of eithersex at any stage of development. In some embodiments, “animal” refers tonon-human animals at any stage of development. In certain embodiments,the nonhuman animal is a mammal (e.g., a rodent, a mouse, a rat, arabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig).In some embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and worms. In some embodiments, theanimal is a transgenic animal, genetically-engineered animal, or aclone. In some embodiments, the animal is a transgenic non-human animal,genetically-engineered non-human animal, or a non-human clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more entities, for example, moieties, molecules,and/or ARMMs, means that the entities are physically associated orconnected with one another, either directly or via one or moreadditional moieties that serves as a linker, to form a structure that issufficiently stable so that the entities remain physically associatedunder the conditions in which the structure is used, e.g., physiologicalconditions. An ARMM is typically associated with an agent, for example,a nucleic acid, protein, or small molecule, by a mechanism that involvesa covalent or non-covalent association. In certain embodiments, theagent to be delivered is covalently bound to a molecule that is part ofthe ARMM, for example, an ARRCD1 protein or fragment thereof, a TSG101protein or fragment thereof, or a lipid or protein that forms part ofthe lipid bilayer of the ARMM. In some embodiments, a peptide or proteinis associated with an ARRCD1 protein or fragment thereof, a TSG101protein or fragment thereof, or a lipid bilayer-associated protein by acovalent bond (e.g., an amide bond). In some embodiments, theassociation is via a linker, for example, a cleavable linker. In someembodiments, an entity is associated with an ARMM by inclusion in theARMM, for example, by encapsulation of an entity (e.g., an agent) withinthe ARMM. For example, in some embodiments, an agent present in thecytoplasm of an ARMM-producing cell is associated with an ARMM byencapsulation of agent-comprising cytoplasm in the ARMM upon ARMMbudding. Similarly, a membrane protein, or other molecule associatedwith the cell membrane of an ARMM producing cell may be associated withan ARMM produced by the cell by inclusion into the ARMM membrane uponbudding.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments,where a nucleic acid is biologically active, a portion of that nucleicacid that shares at least one biological activity of the whole nucleicacid is typically referred to as a “biologically active” portion.

Biomarker: The term “biomarker” as used herein in the context ofARMM-based. diagnostics, refers to a detectable molecule (e.g., aprotein, a peptide, a proteoglycan, a glycoprotein, a lipoprotein, acarbohydrate, a lipid, a nucleic acid (e.g., DNA, such as cDNA oramplified DNA, or RNA, such as mRNA), an organic or inorganic chemical,a small molecule (e.g., second messenger, a metabolite), or adiscriminating molecule or discriminating fragment of any of theforegoing), that is present in or derived from a biological samplecontaining ARMMs, or any ratio of such molecules, or any othercharacteristic that is objectively measured and evaluated as anindicator of a specific biologic feature or process, for example, ofcell or vesicle identity, of a normal or a pathogenic processes, or apharmacologic response to a therapeutic intervention, or an indicationthereof. See Atkinson, A. J., et al., Biomarkers and SurrogateEndpoints: Preferred Definitions and Conceptual Framework, ClinicalPharm. & Therapeutics, 2001 March; 69(3): 89-95. In this context, theterm “derived from” refers to a compound that, when detected, isindicative of a particular molecule being present in the biologicalsample. For example, detection of a particular cDNA can be indicative ofthe presence of a particular RNA transcript or protein in the biologicalsample. As another example, detection of or binding to a particularantibody can be indicative of the presence of a particular antigen(e.g., protein) in the biological sample. In some embodiments, adiscriminating molecule or fragment is a molecule or fragment that, whendetected, indicates presence or abundance of an above-identifiedcompound. A biomarker can, for example, be isolated from an ARMM,directly measured as part of an ARMM, or detected in or determined to beincluded in an ARMM. In some embodiments, the amount of ARMMs detectedin a sample from a subject or in a cell population derived from a sampleobtained from a subject, or the rate of ARMM generation within a sampleor cell population obtained from a subject serves as a biomarker.Methods for the detection of biomarkers are known to those of skill inthe art and include nucleic acid detection methods, protein detectionmethods, carbohydrate detection methods, antigen detection methods, andother suitable methods.

A “biomarker profile” comprises one or more biomarkers (e.g., an mRNAmolecule, a cDNA molecule, a protein, and/or a carbohydrate, or anindication thereof). The biomarkers of the biomarker profile can be inthe same or different classes, such as, for example, a nucleic acid, acarbohydrate, a metabolite, and a protein. A biomarker profile maycomprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, or more biomarkers. In some embodiments, abiomarker profile comprises hundreds, or even thousands, of biomarkers.A biomarker profile can further comprise one or more controls orinternal standards. In some embodiments, the biomarker profile comprisesat least one biomarker that serves as an internal standard. In someembodiments, the presence or level of ARRDC1 or TSG101. in a sample orcell population is used as an internal standard. Biomarker profiles forseveral conditions, diseases, pathologies, and also for normal statesare known to those of skill in the art, and the invention is not limitedto any particular biomarker profile. In some embodiments, the biomarkerprofile used in the context of ARMM-based diagnostic methods asdescribed herein is a biomarker profile that has been described to beuseful for exosome-base diagnostics. Exosomal biomarker profiles areknown to those of skill in the art and biomarker profiles useful for thediagnosis of various disease, including different cancers, stroke,autism, and other diseases, have been described, for example, in U.S.patent application Ser. No. 13/009,285, filed on Jan. 19, 2011(published as US 2011/0151460 A1) by Kaas et al. and entitled MethodsAnd Systems of Using Exosomes For Determining Phenotypes, the entirecontents of which are incorporated herein by reference.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or amino acid sequence,respectively, that are those that occur unaltered in the same positionof two or more related sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences. In some embodiments, two or more sequencesare said to be “completely conserved” if they are 100% identical to oneanother. In some embodiments, two or more sequences are said to be“highly conserved” if they are at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“highly conserved” if they are about 70% identical, about 80% identical,about 90% identical, about 95%, about 98%, or about 99% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are at least 30% identical, at least 40% identical,at least 50% identical, at least 60% identical, at least 70% identical,at least 80% identical, at least 90% identical, or at least 95%identical to one another. In some embodiments, two or more sequences aresaid to be “conserved” if they are about 30% identical, about 40%identical, about 50% identical, about 60% identical, about 70%identical, about 80% identical, about 90% identical, about 95%identical, about 98% identical, or about 99%) identical to one another.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized or useful.

Fusion protein: As used herein, a “fusion protein” includes a firstprotein moiety, e.g., an ARRCD1 protein or fragment thereof, or a TSG101protein or fragment thereof, having a peptide linkage with a secondprotein moiety, for example, a protein to be delivered to a target cell.In certain embodiments, the fusion protein is encoded by a single fusiongene.

Gene: As used herein, the term “gene” has its meaning as understood inthe art. It will be appreciated by those of ordinary skill in the artthat the term “gene” may include gene regulatory sequences promoters,enhancers, etc.) and/or intron sequences. It will further be appreciatedthat definitions of gene include references to nucleic acids that do notencode proteins but rather encode functional RNA molecules such as RNAiagents, ribozymes, tRNAs, etc. For the purpose of clarity it should benoted that, as used in the present application, the term “gene”generally refers to a portion of a nucleic acid that encodes a protein;the term may optionally encompass regulatory sequences, as will be clearfrom context to those of ordinary skill in the art. This definition isnot intended to exclude application of the term “gene” tonon-protein-coding expression units but rather to clarify that, in mostcases, the term as used in this document refers to a protein-codingnucleic acid.

Gene product or expression product: As used herein, the term “geneproduct” or “expression product” generally refers to an RNA transcribedfrom the gene (pre-and/or post-processing) or a polypeptide (pre- and/orpost-modification) encoded by an RNA transcribed from the gene.

Green fluorescent protein: As used herein, the term “green fluorescentprotein” (GFP) refers to a protein originally isolated from thejellyfish Aequorea victoria that fluoresces green when exposed to bluelight or a derivative of such a protein (e.g., an enhanced orwavelength-shifted version of the protein). The amino acid sequence ofwild type CiFP is as follows:

(SEQ ID NO: 1) MENGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYGKLTLKFICTT GKLPVPWPTL VTTFSYGVQC FSRYPDHMKQHDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLVNRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNGIKVNFKIRHN IEDGSVQLAD HYQQNIPIGD GPVLLPDNHYLSTQSALSKD PNEKRDHMVL LEFVTAAGIT HGMDELYKProteins that are at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99%homologous are also considered to be green fluorescent proteins.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e,g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% similar. The term “homologous” necessarily refers to acomparison between at least two sequences (nucleotides sequences oramino acid sequences). In accordance with the invention, two nucleotidesequences are considered to be homologous if the polypeptides theyencode are at least about 50% identical, at least about 60% identical,at least about 70% identical, at least about 80% identical, or at leastabout 90% identical for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous nucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. Both the identity and the approximatespacing of these amino acids relative to one another must be consideredfor nucleotide sequences to be considered homologous. For nucleotidesequences less than 60 nucleotides in length, homology is determined bythe ability to encode a stretch of at least 4-5 uniquely specified aminoacids. In accordance with the invention, two protein sequences areconsidered to be homologous if the proteins are at least about 50%identical, at least about 60% identical, at least about 70% identical,at least about 80% identical, or at least about 90% identical for atleast one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twonucleic acid sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: :Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana. Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux. J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs, Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. et al. J. Molec.Biol., 215, 403 (1990)),

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of gene expression may be determinedusing standard techniques for measuring mRNA and/or protein levels.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been (1) separated from at least some of the componentswith which it was associated when initially produced (whether in natureor in an experimental setting), and/or (2) produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. In someembodiments, isolated substances are more than about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or more than about 99% pure, Asused herein, a substance is “pure” if it is substantially free of othercomponents.

microRNA (miRNA): As used herein, the term “microRNA” or “miRNA” refersto an RNAi agent that is approximately 21 nucleotides (nt)-23 nt inlength. miRNAs can range between 18 nt-26 nt in length. Typically,miRNAs are single-stranded. However, in some embodiments, miRNAs may beat least partially double-stranded. In certain embodiments, miRNAs maycomprise an RNA duplex (referred to herein as a “duplex region”) and mayoptionally further comprises one to three single-stranded overhangs. Insome embodiments, an RNAi agent comprises a duplex region ranging from15 by to 29 by in length and optionally further comprising one or twosingle-stranded overhangs. An miRNA may be formed from two RNA moleculesthat hybridize together, or may alternatively be generated from a singleRNA molecule that includes a self-hybridizing portion. In general, free5′ ends of miRNA molecules have phosphate groups, and free 3′ ends havehydroxyl groups. The duplex portion of an miRNA usually, but does notnecessarily, comprise one or more bulges consisting of one or moreunpaired nucleotides. One strand of an miRNA includes a portion thathybridizes with a target RNA. In certain embodiments, one strand of themiRNA is not precisely complementary with a region of the target RNA,meaning that the miRNA hybridizes to the target RNA with one or moremismatches. In some embodiments, one strand of the miRNA is preciselycomplementary with a region of the target RNA, meaning that the miRNAhybridizes to the target RNA with no mismatches. Typically, miRNAs arethought to mediate inhibition of gene expression by inhibitingtranslation of target transcripts. However, in some embodiments, miRNAsmay mediate inhibition of gene expression by causing degradation oftarget transcripts.

The term “microvesicle,” as used herein, refers to a droplet of liquidsurrounded by a lipid bilayer. In some embodiments, a microvesicle has adiameter of about 10 nm to about 1000 nm. In some embodiments, amicrovesicle has a diameter of at least about 10 nm, at least about 20nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, atleast about 60 nm, at least about 70 nm, at least about 80 nm, at leastabout 90 nm, at least about 100 nm, at least about 125 nm, at leastabout 150 nm, at least about 175 nm, at least about 200 nm, at leastabout 250 nm, at least about 300 nm, at least about 400 nm, or at leastabout 500 nm. In some embodiments, a microvesicle has a diameter of lessthan about 1000 nm, less than about 900 nm, less than about 800 nm, lessthan about 700 nm, less than about 600 nm, lesson about 500 nm, lessthan about 400 nm, less than about 300 nm, less than about 250 nm, lessthan about 200 nm, lesson about 150 nm, less than about 100 nm, lessthan about 90 nm, less than about 80 nm, lesson about 70 nm, lessonabout 60 nm, or less than about 50 nm. The term microvesicle includesmicrovesicle shed from cells as well as synthetically producedmicrovesicles. Microvesicles shed from cells typically comprise theantigenic content of the cells from which they originate. Microvesiclesshed from cells also typically comprise an asymmetric distribution ofphospholipids, reflecting the phospholipid distribution of the cellsfrom which they originate. In some embodiments, the inner membrane ofmicrovesicles provided herein, e.g., of some ARMMs, comprises themajority of aminophospholipids, phosphatidylserine, and/orphosphatidylethanolamine within the lipid bilayer.

The term “ARMM,” as used herein, refers to a microvesicle comprising anARRDC1 protein or fragment thereof, and/or TSG101 protein or fragmentthereof. The molecular mechanism by which ARMMs are produced isdescribed in more detail elsewhere herein. In some embodiments, the ARMMis shed from a cell, and comprises a molecule, for example, a nucleicacid, protein, or small molecule, present in the cytoplasm of the cell.In some embodiments, the ARMM is shed from a transgenic cell comprisinga recombinant expression construct that includes the transgene, and theARMM comprises a gene product, for example, a transcript or a proteinencoded by the expression construct. In some embodiments, the ARMM isproduced synthetically, for example, by contacting lipid bilayer withinARRDC1 protein in a cell free system in the presence of TSG101, HECTdomain ligase, and VPS4a. In some embodiments, an ARMM lacks a lateendosomal marker. Some ARMMs as provided herein do not include, or arenegative for, one or more exosomal biomarker. Exosomal biomarkers areknown to those of skill in the art and include, but are not limited toCD63, Lamp-1, Lamp-2, CD9, HSPA8, GAPDH, CD81, SDCBP, PDCD6IP, ENO1,ANXA2, ACTB, YWHAZ, HSP90AA1, ANXA5, EEF1A1, YWHAE, PPIA, MSN, CFL1,ALDOA, PGK1, ANXA1, PKM2, HLA-DRA, and YWHAB. For example, some ARMMsprovided herein lack CD63, some ARMMs lack LAMP1, some ARMMs lack CD9,some ARMMs lack CD81, some ARMMs lack CD63 and Lamp-1, some ARMMs lackCD63, Lamp-1, and CD9, some ARMMs lack CD63, Lamp-1, CD81 and CD9, andso forth. Certain ARMMs provided herein may include an exosomalbiomarker. Accordingly, some ARMMs may be negative for one or moreexosomal biomarker, but positive for one or more different exosomalbiomarker. For example, such an ARMM may be negative for CD63 andLamp-1, but may include PGK1 or GAPDH; or may be negative for CD63,Lamp-1, CD9 and CD81, but may be positive for HLA-DRA. In someembodiments, ARMMs include an exosomal biomarker, but at a lower levelthan a level found in exosomes. For example, some ARMMs include one ormore exosomal biomarkers at a level of less than about 1%, less thanabout 5%, less than about 10%, less than about 20%, less than about 30%,less than about 40%, or less than about 50% of the level of thatbiomarker found in exosomes. To give a non-limiting example, in someembodiments, an ARMM may be negative for CD63 and Lamp-1, include CD9 ata level of less than about 5% of the level of CD9 typically found inexosomes, and be positive for ACTB. Exosomal biomarkers in addition tothose listed above are known to those of skill in the art, and theinvention is not limited in this regard.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into an oligonucleotide chain. In some embodiments, anucleic acid is a compound and/or substance that is or can beincorporated into an oligonucleotide chain via a phosphodiester linkage.In some embodiments, “nucleic acid” refers to individual nucleic acidresidues (e.g. nucleotides and/or nucleosides). In some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising individualnucleic acid residues. As used herein, the terms “oligonucleotide” and“polynucleotide” can be used interchangeably to refer to a polymer ofnucleotides (e.g., a string of at least two nucleotides). In someembodiments, “nucleic acid” encompasses RNA as well as single and/ordouble-stranded DNA and/or cDNA. Furthermore, the terms “nucleic acid,”“DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e.analogs having other than a phosphodiester backbone. For example, theso-called “peptide nucleic acids,” which are known in the art and havepeptide bonds instead of phosphodiester bonds in the backbone, areconsidered within the scope of the present invention. The term“nucleotide sequence encoding an amino acid sequence” includes allnucleotide sequences that are degenerate versions of each other and/orencode the same amino acid sequence. Nucleotide sequences that encodeproteins and/or RNA may include introns. Nucleic acids can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, nucleic acids cancomprise nucleoside analogs such as analogs having chemically modifiedbases or sugars, backbone modifications, etc. A nucleic acid sequence ispresented in the 5′ to 3′ direction unless otherwise indicated. The term“nucleic acid segment” is used herein to refer to a nucleic acidsequence that is a portion of a longer nucleic acid sequence. In manyembodiments, a nucleic acid segment comprises at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, ormore residues. In some embodiments, a nucleic acid is or comprisesnatural nucleosides (e adenosine, thymidine, guanosine, cytidine,uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine,8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine);chemically modified bases; biologically modified bases (e.g., methylatedbases); intercalated bases; modified sugars (e.g., 2′-fluororibose,ribose, 2′-deoxyribose, arabinose, and hexose); and/or modifiedphosphate groups (e.g., phosphorothioates and 5′-N-phosphoramiditelinkages). In some embodiments, the present invention is specificallydirected to “unmodified nucleic acids,” meaning nucleic acids (e.g.polynucleotides and residues, including nucleotides and/or nucleosides)that have not been chemically modified in order to facilitate or achievedelivery.

Protein: The term “protein” is used herein interchangeably with theterms polypeptide and peptide, and refers to a polypeptide (i.e., astring of at least two amino acids linked to one another by peptidebonds). Proteins may include moieties other than amino acids (e.g., maybe glycoproteins) and/or may be otherwise processed or modified. Thoseof ordinary skill in the art will appreciate that a “protein” can be acomplete polypeptide chain as produced by a cell (with or without asignal sequence), or can be a functional portion thereof. Those ofordinary skill will further appreciate that a protein can sometimesinclude more than one polypeptide chain, for example linked by one ormore disulfide bonds or associated by other means. Polypeptides maycontain L-amino acids, D-amino acids, or both and may contain any of avariety of amino acid modifications or analogs known in the art. Usefulmodifications include, e.g., addition of a chemical entity such as acarbohydrate group, a phosphate group, a farnesyl group, an isofarnesylgroup, a fatty acid group, an amide group, a terminal acetyl group, alinker for conjugation, functionalization, or other modification (e,g.,alpha amidation), etc. In certain embodiments, the modifications of thepeptide lead to a more stable peptide (e.g., greater half-life in vivo).These modifications may include cyclization of the peptide, theincorporation of D-amino acids, etc. None of the modifications shouldsubstantially interfere with the desired biological activity of thepeptide. In certain embodiments, the modifications of the peptide lead.to a more biologically active peptide. In some embodiments, polypeptidesmay comprise natural amino acids, non-natural amino acids, syntheticamino acids, amino acid analogs, and combinations thereof.

Reprogramming factor: As used herein, the term “reprogramming factor”refers to a factor that, alone or in combination with other factors, canchange the state of a cell from a somatic, differentiated state into apluripotent stem cell state. Non-limiting examples of reprogrammingfactors include a protein (e.g., a transcription factor), a peptide, anucleic acid, or a small molecule. Known reprogramming factors that areuseful for cell reprogramming include, but are not limited to Oct4,Sox2, Klf4, and c-myc. Similarly, a programming factor may be used tomodulate cell differentiation, for example, to facilitate or induce celldifferentiation towards a desired lineage.

RNA interference (RNAi): As used herein, the term “RNA interference” or“RNAi” refers to sequence-specific inhibition of gene expression and/orreduction in target RNA levels mediated by an RNA, which RNA comprises aportion that is substantially complementary to a target RNA. Typically,at least part of the substantially complementary portion is within thedouble stranded region of the RNA. In some embodiments, RNAi can occurvia selective intracellular degradation of RNA. In some embodiments,RNAi can occur by translational repression.

RNAi agent: As used herein, the term “RNAi agent” or “RNAi” refers to anRNA, optionally including one or more nucleotide analogs ormodifications, having a structure characteristic of molecules that canmediate inhibition of gene expression through an RNAi mechanism. In someembodiments. RNAi agents mediate inhibition of gene expression bycausing degradation of target transcripts. In some embodiments, RNAiagents mediate inhibition of gene expression by inhibiting translationof target transcripts. Generally, an RNAi agent includes a portion thatis substantially complementary to a target RNA. In some embodiments,RNAi agents are at least partly double-stranded. In some embodiments,RNAi agents are single-stranded. In some embodiments, exemplary RNAiagents can include siRNA, shRNA, and/or miRNA. In some embodiments, RNAiagents may be composed entirely of natural RNA nucleotides (i.e.,adenine, guanine, cytosine, and uracil). In some embodiments, RNAiagents may include one or more non-natural RNA nucleotides (e.g.,nucleotide analogs, DNA nucleotides, etc.). Inclusion of non-natural RNAnucleic acid residues may be used to make the RNAi agent more resistantto cellular degradation than RNA. In some embodiments, the term “RNAiagent” may refer to any RNA, RNA derivative, and/or nucleic acidencoding an RNA that induces an RNAi effect (e.g., degradation of targetRNA and/or inhibition of translation). In some embodiments, an RNAiagent may comprise a blunt-ended (i.e., without overhangs) dsRNA thatcan act as a Dicer substrate. For example, such an RNAi agent maycomprise a blunt-ended dsRNA which is ≥25 base pairs length, which mayoptionally be chemically modified to abrogate an immune response.

RNAi-inducing agent: As used herein, the term “RNAi-inducing agent”encompasses any entity that delivers, regulates, and/or modifies theactivity of an RNAi agent. In some embodiments, RNAi-inducing agents mayinclude vectors (other than naturally occurring molecules not modifiedby the hand of man) whose presence within a cell results in RNAi andleads to reduced expression of a transcript to which the RNAi-inducingagent is targeted. In some embodiments, RNAi-inducing agents areRNAi-inducing vectors. In some embodiments, RNAi-inducing agents arecompositions comprising RNAi agents and one or more pharmaceuticallyacceptable excipients and/or carriers. In some embodiments, anRNAi-inducing agent is an “RNAi-inducing vector,” which refers to avector whose presence within a cell results in production of one or moreRNAs that self-hybridize or hybridize to each other to form an RNAiagent (e.g. siRNA, shRNA, and/or miRNA). In various embodiments, thisterm encompasses plasmids, e.g., DNA vectors (whose sequence maycomprise sequence elements derived from a virus or viruses (other thannaturally occurring viruses or plasmids that have not been modified bythe hand of man), whose presence within a cell results in production ofone or more RNAs that self-hybridize or hybridize to each other to forman RNAi agent. In general, the vector comprises a nucleic acid operablylinked to expression signal(s) so that one or more RNAs that hybridizeor self-hybridize to form an RNAi agent are transcribed when the vectoris present within a cell. Thus the vector provides a template forintracellular synthesis of the RNA or RNAs or precursors thereof. Forpurposes of inducing RNAi, presence of a viral genome in a cell (e.g.,following fusion of the viral envelope with the cell membrane) isconsidered sufficient to constitute presence of the virus within thecell. In addition, for purposes of inducing RNAi, a vector is consideredto be present within a cell if it is introduced into the cell, entersthe cell, or is inherited from a parental cell, regardless of whether itis subsequently modified or processed within the cell. An RNAi-inducingvector is considered to be targeted to a transcript if presence of thevector within a cell results in production of one or more RNAs thathybridize to each other or self-hybridize to form an RNAi agent that istargeted to the transcript, i.e., if presence of the vector within acell results in production of one or more RNAi agents targeted to thetranscript.

Short, interfering RNA (siRNA): As used herein, the term “short,interfering RNA” or “siRNA” refers to an RNAi agent comprising an RNAduplex (referred to herein as a “duplex region”) that is approximately19 base pairs (bp) in length and optionally further comprises one tothree single-stranded overhangs. In some embodiments, an RNAi agentcomprises a duplex region ranging from 15 by to 29 by in length andoptionally further comprising one or two single-stranded overhangs. AnsiRNA may be formed from two RNA molecules that hybridize together, ormay alternatively be generated from a single RNA molecule that includesa self-hybridizing portion. In general, free 5′-ends of siRNA moleculeshave phosphate groups, and free 3′-ends have hydroxyl groups. The duplexportion of an siRNA may, but typically does not, comprise one or morebulges consisting of one or more unpaired nucleotides. One strand of ansiRNA includes a portion that hybridizes with a target transcript. Incertain embodiments, one strand of the siRNA is precisely complementarywith a region of the target transcript, meaning that the siRNAhybridizes to the target transcript without a single mismatch. In someembodiments, one or more mismatches between the siRNA and the targetedportion of the target transcript may exist. In some embodiments in whichperfect complementarity is not achieved, any mismatches are generallylocated at or near the siRNA termini. In some embodiments, siRNAsmediate inhibition of gene expression by causing degradation of targettranscripts.

Short hairpin RNA (shRNA): As used herein, the term “short hairpin RNA”or “shRNA” refers to an RNAi agent comprising an RNA having at least twocomplementary portions hybridized or capable of hybridizing to form adouble-stranded (duplex) structure sufficiently long to mediate RNAi(typically at least approximately 19 hp in length), and at least onesingle-stranded portion, typically ranging between approximately 1nucleotide (nt) and approximately 10 nt in length that forms a loop. Insome embodiments, an shRNA comprises a duplex portion ranging from 15 byto 29 bp in length and at least one single-stranded portion, typicallyranging between approximately 1 nt and approximately 10 nt in lengththat forms a loop. The duplex portion may, but typically does not,comprise one or more bulges consisting of one or more unpairednucleotides. In some embodiments, siRNAs mediate inhibition of geneexpression by causing degradation of target transcripts. shRNAs arethought to be processed into siRNAs by the conserved cellular RNAimachinery. Thus shRNAs may be precursors of siRNAs. Regardless, siRNASin general are capable of inhibiting expression of a target RNA, similarto siRNAs.

Small molecule: In general, a “small molecule” refers to a substantiallynon-peptidic, non-oligomeric organic compound either prepared in thelaboratory or found in nature. Small molecules, as used herein, canrefer to compounds that are “natural product-like,” however, the term“small molecule” is not limited to “natural product-like” compounds.Rather, a small molecule is typically characterized in that it containsseveral carbon-carbon bonds, and has a molecular weight of less than1500 g/mol, less than 1250 g/mol, less than 1000 g/mol, less than 750g/mol, less than 500 g/mol, or less than 250 g/mol, although thischaracterization is not intended to be limiting for the purposes of thepresent invention. In certain other embodiments, natural-product-likesmall molecules are utilized.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g, for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g, nucleic acid, drug, therapeutic agent, diagnostic agent,prophylactic agent, etc.) that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, improve symptoms of, diagnose, prevent, and/ordelay the onset of the disease, disorder, and/or condition.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particulardisease, disorder, and/or condition. For example, “treating” cancer mayrefer to inhibiting survival, growth, and/or spread of a tumor.Treatment may be administered to a subject who does not exhibit signs ofa disease, disorder, and/or condition and/or to a subject who exhibitsonly early signs of a disease, disorder, and/or condition for thepurpose of decreasing the risk of developing pathology associated withthe disease, disorder, and/or condition.

Vector: As used herein, “vector” refers to a nucleic acid molecule whichcan transport another nucleic acid to which it has been linked. In someembodiment, vectors can achieve extra-chromosomal replication and/orexpression of nucleic acids to which they are linked in a host cell suchas a eukaryotic and/or prokaryotic cell. Vectors capable of directingthe expression of operatively linked genes are referred to herein as“expression vectors.”

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The plasma membrane is a dynamic structure that can bud inward oroutward to generate membrane-encapsulated vesicles (1, 2). Inwardbudding leads to the formation of endosomes, which can mediate thesorting and degradation of receptors and other plasma membrane proteinsby a highly conserved ESCRT (Endosomal Sorting Complex Required forTransport) machinery that recognizes and guides ubiquitinated receptorsfrom plasma membranes to late endosomes and then to lysosomes, where thereceptors are degraded (for reviews, see refs (1, 3, 4)). Tumorsusceptibility gene 101 (TSG101), an essential ESCRT component, iscentral to endosomal sorting (5, 6). Using a unique ubiquitin enzymevariant (UEV) domain that binds to ubiquitin and recognizes thetetrapeptide protein motif, PS/TAP (7-9), TSG101 interacts with theearly endosomal protein Hrs (6, 10, 11) and late endosome-associatedAlix (12). to mediate the trafficking of ubiquitinated receptors fromearly endosomes to late endosomes; there, additional ESCRT proteins andan ATPase (VPS4) drive the formation of, and delivery of endosomal cargoto, endosomal-membrane-derived vesicles known as multivesicular bodies(MVBs) (3, 13). Fusion of MVBs with lysosomes leads to degradation ofthe cargo, whereas trafficking and fusion of MVB-containing lateendosomes with the plasma membrane deliver small vesicles termed“exosomes” into the extracellular space (2, 14, 15).

Outward budding of plasma membranes occurs during egress of buddingviruses from cells. The best characterized example of viral budding,which hijacks the ESCRT machinery, involves the human immunodeficiencyvirus (HIV) (16-18). HIV budding is driven by the viral Gag protein,which localizes to the plasma membrane of host cells. In the “latedomain” of HIV Gag is a critical PTAP motif that interacts with the LEVdomain of TSG101 (19, 20), enabling Gag to recruit TSG101 and otherESCRT proteins, which normally reside on endosomal membranes, to thecell surface (12, 21, 22). By such recruitment, Gag can initiate plasmamembrane budding. Disruption of the Gag/TSG101 interaction througheither mutation of the PTAP motif in Gag or overexpression of mutantTSG101 can block the release of WY viral particles (23-25). Many otherviruses also recruit TSG101 or other ESCRT components to aid theirbudding at the cell surface (26, 27).

While viral budding and MVB formation are topologically similar in thatboth processes utilize the ESCRT machinery and involve the budding ofmembranes away from the cytosol, the two events are in fact functionallyand structurally distinct. MVBs are formed by invagination of lateendosome limiting membranes into the lumen of the organelle, whereasbudding of viruses normally occurs at the cell surface and results inrelease of viral particles from the cytoplasm extracellularly(evagination). Thus, the analogy between MVB formation and viral budding(18, 26) is incomplete.

Some aspects of this invention relate to the discovery of an intrinsiccellular process, termed DPMB (direct plasma membrane budding) thatunderlies MVB-independent vesicle formation at the cell surface. Asdescribed in more detail elsewhere herein, DPMB involves the recruitmentof TSG101 to the cell surface by the ARRDC1protein and ismechanistically distinct from MVB formation and from MVB-related exosomeformation, and leads directly to evagination of the plasma membrane andthe release of a novel type of microvesicle, termed ARMM, into theextracellular space.

Microvesicles

Some aspects of this invention provide isolated arrestindomain-containing protein 1 (ARIWC1)-mediated microvesicles (ARMMs).Such ARMMs typically include a lipid bilayer and an ARRDC1 protein orfragment thereof. In some embodiments, an ARMM further includes aTSG101. protein or fragment thereof. In some embodiments, an ARMMincludes additional or different markers, for example, proteins andmolecules associated with the cellular ESCRT machinery.

ARRDC1

ARRDC1 is a protein that comprises a PSAP and a PPXY motif, alsoreferred to herein as a PSAP and PPXY domain, respectively, in itsC-terminus, and interacts with TSG101 as shown herein. Exemplary,nonlimiting ARRDC1 protein sequences are provided herein, andadditional, suitable ARRDC1 protein sequences, isoforms, and fragmentsaccording to aspects of this invention are known in the art. It will beappreciated by those of skill in the art that this invention is notlimited in this respect. Exemplary ARRDC1 sequences include thefollowing (PSAP and PPXY motifs are marked):

>gi|22748653|ref|NP_689498.1|arrestindomain-containing protein 1 [Homo sapiens] (SEQ ID NO: 2)MGRVQLFEISLSHGRVVYSPGEPLAGTVR VRLGAPLPFRAIRVTCIGSCGVSNKANDTAWVVEEGYFNSSLSLADKGSLPAGEHSFPF QFLLPATAPTSFEGPFGKIVHQVRAAIHTPRFSKDHKCSLVFYILSPLNLNSIPDIEQPNVA SATKKFSYKLVKTGSVVLTASTDLRGYVVGQALQLHADVENQSGKDTSPVVASLLQKVS YKAKRWIHDVRTIAEVEGAGVKAWRRAQWHEQILVPALPQSALPGCSLIHIDYYLQVSLKAP EATVTLPVFIGNIAVNHAPVSPRPGLG

LDPVFLSTKSHSQRQPLLATLSSVPGAPEPC PQDGSPASHPLHPPLCISTGATVPYFAEG

CGGVEPSLTPES >gi|244798004|ref|NP_001155957.1|arrestindomain-containing protein 1 isoform a  [Mus musculus] (SEQ ID NO: 3)MGRVQLFEIRLSQGRVVYGPGEPLAG TVHLRLGAPLPFRAIRVTCMGSCGVSTKANDGAWVVEESYFNSSLSLADKGSLPAGEHNFPF QFLLPATAPTSFEGPFGKIVHQVRASIDTPRFSKDHKCSLVFYILSPLNLNSIPDIEQPNVAS TTKKFSYKLVKTGNVVLTASTDLRGYVVGQVLRLQADIENQSGKDTSPVVASLLQKV SYKAKRWIYDVRTIAEVEGTGVKAWRRAQWQEQILVPALPQSALPGCSLIHIDYYLQVSM KAPEATVTLPLFVGNIAVNQTPLSPCPGR

SLSTKSHSQQQPLSAPLGSVSVTTTEPW VQVGSPARHSLHPPLCISIGATVPYFAEGSAG

SCGAAGTDLGLIPGS >gi|244798112|ref|NP_848495.2|arrestindomain-containing protein 1 isoform b  [Mus musculus] (SEQ ID NO: 4)MGRVQLFEIRLSQGRVVYGPGEPLAGTVHL RLGAPLPFRAIRVTCMGSCGVSTKANDGAWVVEESYFNSSLSLADKGSLPAGEHNFPF QFLLPATAPTSFEGPFGKIVHQVRASIDTPRFSKDHKCSLVFYILSPLNLNSIPDIEQPNVAS TTKKFSYKLVKTGNVVLTASTDLRGYVVGQVLRLQADIENQSGKDTSPVVASLLQVSYK AKRWIYDVRTIAEVEGTGVKAWRRAQWQEQILVPALPQSALPGCSLIHIDYYLQVSMK APEATVTLPLFVGNIAVNQTPLSPCPGR

SLSTKSHSQQQPLSAPLGSVSVTTTEPW VQVGSPARHSLHPPLCISIGATVPYFAEGSAG

SCGAAGTDLGLIPGS

TSG101

Tumor susceptibility gene 101, also referred to herein as TSG101, is aprotein encoded by this gene belongs to a group of apparently inactivehomologs of ubiquitin-conjugating enzymes. The protein contains acoiled-coil domain that interacts with stathmin, a cytosolicphosphoprotein implicated in tumorigenesis. TSG101 is a protein thatcomprises a UEV domain, and interacts with ARRDC1 as shown herein.Exemplary, non-limiting TSG101 protein sequences are provided herein,and additional, suitable TSG101 protein sequences, isoforms, andfragments according to aspects of this invention are known in the art.It will be appreciated by those of skill in the art that this inventionis not limited in this respect. Exemplary TSG101 sequences include thefollowing:

>gi|5454140|ref|NP_006283.1| tumorsusceptibility gene 101 protein [Homo sapiens] (SEQ ID NO: 5)MAVSESQLKKMVSKYKYRDLTVRETVNVITLYKDLKPVLDSYVFNDGSSRELMNLTGTIPVPYRGNTYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHEWKHPQSDLLGLIQVMIVVFGDEPPVFSRPISASYPPYQATGPPNTSYMPGMPGGISPYPSGYPPNPSGYPGCPYPPGGPYPATTSSQYPSQPPVTTVGPSRDGTISEDTIRASLISAVSDKLRWRMKEEMDRAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDTIFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY >gi|11230780|ref|NP_068684.1| tumorsusceptibility gene 101 protein [Mus musculus] (SEQ ID NO: 6)MAVSESQLKKMMSKYKYRDLTVRQTVNVIAMYKDLKPVLDSYVFNDGSSRELVNLTGTIPVRYRGNIYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHDWKHPRSELLELIQIMIVIFGEEPPVFSRPTVSASYPPYTATGPPNTSYMPGMPSGISAYPSGYPPNPSGYPGCPYPPAGPYPATTSSQYPSQPPVTTVGPSRDGTISEDTIRASLISAVSDKLRWRMKEEMDGAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDTIFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY >gi|48374087|ref|NP_853659.2| tumorsusceptibility gene 101 protein [Rattus norvegicus] (SEQ ID NO: 7)MAVSESQLKKMMSKYKYRDLTVRQTVRQTVNVIAMYKDLKPVLDSYVFNDGSSRELVNLTGTIPVRYRGNIYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHDWKHPRSELLELIQIMIVIFGEEPPVFSRPTVSASYPPYTAAGPPNTSYLPSMPSGISAYPSGYPPNPSGYPGCPYPPAGPYPATTSSQYPSQPPVTTAGPSRDGTISEDTIRASLISAVSDKLRWRMKEEMDGAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDTIFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY

The UEV domain in these sequences includes amino acids 1-145 (underlinedin the sequences above). The structure of UEV domains is known to thoseof skill in the art (see, e.g, Owen Pornillos et al, Structure andfunctional interactions of the Tsg101 UEV domain, EWBO J. 2002 May 15;21(10): 2397-2406, the entire contents of which are incorporated hereinby reference).

In some embodiments, microvesicle, ARMMs, are provided that comprise anARRDC1 protein fragment, and/or a TSG101 protein fragment. In someembodiments, fusion proteins are provided that comprise an ARRDC1protein fragment and/or a TSG101 protein fragment. In some embodiments,expression construct are provided that encode an ARRDC1 protein fragmentand/or a TSG101 protein fragment. In some embodiments, the ARRDC1protein fragment is a C-terminal ARRDC1protein fragment. In someembodiments, the ARRDC1 protein fragment comprises the PSAP motif and atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, at least 100, at least 110,at least 120, at least 130, at least 140, at least 150, at least 160, atleast 170, at least 180, at least 190, at least 200, at least 210, atleast 220, at least 230, at least 240. at least 250, at least 260, atleast 270, at least 280, at least 290, or at least 300 contiguous aminoacids of the ARRCD1 sequence. In some embodiments, the TSG101 proteinfragment comprises a TSG101 UEV domain. In some embodiments, the TSG101protein fragment comprises the UEV domain and comprises at least 150, atleast 160, at least 170, at least 180, at least 190, at least 200, atleast 210, at least 220, at least 230, at least 240, at least 250, atleast 260, at least 270, at least 280, at least 290, or at least 300contiguous amino acids of the TSG101 sequence.

In some embodiments, the inventive microvesicles, e.g., ARMMs, furthercomprise a cell surface protein, for example, an integrin, a receptortyrosine kinase, a G-protein coupled receptor, or a membrane-boundimmunoglobulin. Other cell surface proteins may also be included in anARMM. Integrins, receptor tyrosine kinases, G-protein coupled receptors,and a membrane-bound immunoglobulins suitable for use with embodimentsof this invention will be apparent to those of skill in the art and theinvention is not limited in this respect. For example, in someembodiments, the integrin is an α1β1, α2β1, α4β1, α5β1, α6β1, αLβ2,αMβ2, αIIbβ3, αVβ3, αVβ5, αVβ6, and α6β4 integrin. In some embodiments,the receptor tyrosine kinase is a an EGF receptor (ErbB family), insulinreceptor, PDGF receptor, FGF receptor, VEGF receptor, HGF receptor, Trkreceptor, Eph receptor, AXL receptor, LTK receptor, TIE receptor, RORreceptor, DDR receptor, RET receptor, KLG receptor, RYK receptor, orMuSK receptor. In some embodiments, the G-protein coupled receptor is arhodopsin-like receptor, the secretin receptor, metabotropicglutamate/pheromone receptor, cyclic AMP receptor, frizzled/smoothenedreceptor, CXCR4, CCR5, or beta-adrenergic receptor.

Some aspects of this invention relate to the recognition that ARMMs aretaken up by target cells, and ARMM uptake results in the release of thecontents of the ARMM into the cytoplasm of the target cells. Someaspects of this invention relate to the recognition that this can beused to deliver an agent in ARMMs to the target cell or a population oftarget cells, for example, by contacting the target cell with ARMMscomprising the agent to be delivered. Accordingly, some aspects of thisinvention provide ARMMs that comprise an agent, for example, arecombinant nucleic acid, a recombinant protein, or a synthetic smallmolecule.

In some embodiments, the agent is an agent that effects a desired changein the target cell, for example, a change in cell survival,proliferation rate, a change in differentiation stage, a change in acell identity, a change in chromatin state, a change in thetranscription rate of one or more genes, a change in the transcriptionalprofile, or a post-transcriptional change in gene compression of thetarget cell. It will be understood by those of skill in the art, thatthe agent to be delivered will be chosen according to the desired effectin the target cell. For example, to effect a change in thedifferentiation stage of a target cell, for example, to reprogram adifferentiated target cell into an embryonic stem cell-like stage, thecell is contacted, in some embodiments, with ARMMs with reprogrammingfactors, for example, Oct4, Sox2, c-Myc, and/or KLF4. Similarly, toeffect the change in the chromatin state of a target cell, the cell iscontacted, in some embodiments, with ARMMs containing a chromatinmodulator, for example, a DNA methyltransferase, or a histonedeacetylase. For another example, if survival of the target cell is tobe diminished, the target cell, in some embodiments, is contacted withARMMs comprising a cytotoxic agent, for example, a chemotherapeuticdrug. Additional agents suitable for inclusion into ARMMs and for aARMM-mediated delivery to a target cell or target cell population willbe apparent to those skilled in the art, and the invention is notlimited in this respect.

In some embodiments, the agent is included in the ARMMs by contactingcells producing the ARMMs with the agent. For example, if the agent is asmall molecule, for example a therapeutic drug to be delivered to atarget cell population within the body of a subject, ARMMs containingthe drug are produced by contacting cells expressing ARRDC1 and TSG101with the drug in an amount and for a time sufficient to generate ARMMscontaining the drug. For another example, if the agent is a nucleic acidor a protein, ARMMs containing nucleic acid or the protein are producedby expressing the nucleic acid or the protein in cells expressing ARRDC1and TSG101, for example, from a recombinant expression construct.

In some embodiments, the agent is conjugated to the ARRDC1 protein, theARRDC1 fragment, the TSG101 protein, or the TSG101 fragment. In someembodiments, where the agent is a protein, the protein may be conjugatedto the ARRDC protein, the ARRDC1 fragment, the TSG101 protein, or theTSG101 fragment, by expressing the protein agent as a fusion with theARRDC1 protein, the ARRDC1 fragment, the TSG101 protein, or the TSG101fragment.

In some embodiments, ARMMs are provided that include a recombinant or asynthetic nucleic acid. Such ARMMs can be used to deliver therecombinant or synthetic nucleic acids to a target cell or target cellpopulation. In some embodiments, the recombinant nucleic acid comprisesan RNA, for example, an RNA encoding a protein (e.g., an mRNA), or anon-coding RNA. In some embodiments, the nucleic acid comprises an RNAiagent, for example, an antisense RNA, a small interfering RNA (siRNA), asmall hairpin RNA (shRNA), a microRNA (miRNA), a small nuclear RNA(snRNA), a small nucleolar RNA (snoRNA), or a long intergenic non-codingRNA (lincRNA), or a precursor thereof. Some embodiments, ARMMs areprovided that include a recombinant structural RNA, a ribozyme, or aprecursor thereof.

Coding RNAs, RNAi agents, structural RNAs, and ribozymes, as well asprecursors thereof, are well known to those skilled in the art andsuitable RNAs and RNAi agents according to aspects of this inventionwill be apparent to the skilled artisan. It will be appreciated that theinvention is not limited in this respect. ARMMs including RNA can beused to express the RNA function in a target cell without the need forgenetic manipulation of the target cell. For example, ARMMs includingprotein-encoding nucleic acids can be used to express the encodedprotein in a target cell or cell population upon ARMMs uptake withoutthe need to genetically manipulate the target cell or cell population.For another example, ARMMs including an RNAi agent can be used to knockdown a gene of interest in the target cell or the target cell populationwithout the need to genetically amended claims department cell or cellpopulation. For a third example, ARMMs including a ribozyme can be usedto modulate the expression of a target nucleic acid, or to edit a targetmRNA and a target cell without the need for genetic manipulation.

In some embodiments, ARMMs are provided that include a DNA, for example,a vector including an expression construct, a LINE sequence, a SINEsequence, a composite SINE sequence, or an LTR-retrotransposon sequence.ARMMs containing DNA allow for the transfer of genes or DNA elementsfrom cell to cell, or, in some embodiments, for the targeted insertionof genes or DNA elements into a target cell or target cell type, forexample a pathological target cell type in a subject. In someembodiments, ARMMs are provided that include a DNA encoding a protein.In some embodiments, ARMMs are provided that include a DNA encoding anon-coding RNA, for example, an anti sense RNA, a small interfering RNA(siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a smallnuclear RNA (snRNA), a small nucleolar RNA (snoRNA), or a longintergenic non-coding RNA (lincRNA), or a precursor thereof. In someembodiments, the use of ARMMs containing a DNA has the advantage that ahigher level of expression or a more sustained expression of the encodedprotein or RNA can be achieved in a target cell as compared to directdelivery of the protein or RNA. In some embodiments, the DNA included inthe ARMMs comprises a cell type specific promoter controlling theconscription of the encoded protein or RNA. The use of a cell typespecific promoter allows for the targeted expression of the proteinswere RNA encoded by the ARMM-delivered DNA, which can be used, forexample in some therapeutic embodiments, to minimize the effect onsubpopulations that are not targeted but may take up ARMMs,

in some embodiments, ARMMs are provided that include a detectable label.Such ARMMs allow for the labeling of a target cell without geneticmanipulation. Detectable labels suitable for direct delivery to targetcells are known in the art, and include, but are not limited to,fluorescent proteins, fluorescent dyes, membrane-bound dyes, andenzymes, for example, membrane-bound enzymes, catalyzing the reactionresulting in a detectable reaction product. Detectable labels suitableaccording to some aspects of this invention further includemembrane-bound antigens, for example, membrane-bound ligands that can bedetected with commonly available antibodies or antigen binding agents.

In some embodiments, ARMMs are provided that comprise a therapeuticagent. It will be appreciated, that any therapeutic agent that can beintroduced into a cell shedding ARMMs or that can be packaged intosynthetic ARMMs is suitable for inclusion into ARMMs according to someaspects of this invention. Suitable therapeutic agents include, but arenot limited to, small organic molecules, also referred to as smallmolecules, or small compounds, and biologics, for example, therapeuticproteins, or protein fragments. Some non-limiting examples oftherapeutic agents suitable for inclusion in ARMMs include antibacterialagents, antifungal antibiotics, antimyobacterials, neuraminidaseinhibitors, antineoplastic agents, cytotoxic agents, cholinergic agents,parasympathomimetics, anticholinergic agents, antidepressants,antipsychotics, respiratory and cerebral stimulants, proton pumpinhibitors, hormones and synthetic substitutes, receptor ligands, kinaseinhibitors, chemotherapeutic agents, signaling molecules, kinases,phosphatases, proteases, RNA editing enzymes, nucleases, and zinc fingerproteins.

In some embodiments, ARMMs are provided that comprise a protein to bedelivered to a target cell. In some embodiments, the protein is orcomprises a transcription factor, a transcriptional repressor, afluorescent protein, a kinase, a phosphatase, a protease, a ligase, achromatin modulator, or a recombinase. In some embodiments, the proteinis a therapeutic protein. In some embodiments the protein is a proteinthat effects a change in the state or identity of a target cell. Forexample, in some embodiments, the protein is a reprogramming factor.Suitable transcription factors, transcriptional repressors, fluorescentproteins, kinases, phosphatases, proteases, ligases, chromatinmodulators, recombinases, and reprogramming factors are known to thoseskilled in the art, and the invention is not limited in this respect.

In some embodiments, ARMMs are provided that comprise an agent, forexample, a small molecule, a nucleic acid, or a protein, that iscovalently or non-covalently bound, or conjugated, to an ARRDC1 proteinor fragment thereof, or a TSG101 protein or fragment thereof. In someembodiments, agent is conjugated to the ARRDC1 protein or fragmentthereof, or the TSG101 protein or fragment thereof, via a linker. Thelinker may be cleavable or uncleavable. In some embodiments, the linkercomprises an amide, ester, ether, carbon-carbon, or disulfide bond,although any covalent bond in the chemical art may be used. In someembodiments, the linker comprises a labile bond, cleavage of whichresults in separation of the supercharged protein from the peptide orprotein to be delivered. In some embodiments, the linker is cleavedunder conditions found in the target cell (e.g., a specific pH, areductive environment, or the presence of a cellular enzyme). In someembodiments, the linker is cleaved by a cellular enzyme. In someembodiments, the cellular enzyme is a cellular protease or a cellularesterase. In some embodiments, the cellular protease is a cytoplasmicprotease, an endosomal protease, or an endosomal esterase. In someembodiments, the cellular enzyme is specifically expressed in a targetcell or cell type, resulting in preferential or specific release of thefunctional protein or peptide in the target cell or cell type. Thetarget sequence of the protease may be engineered into the linkerbetween the agent to be delivered and the ARRDC1 protein or the TSG101protein or fragment thereof. In some embodiments, the target cell orcell type is a cancer cell or cancer cell type, a cell or cell type ofthe immune system, or a pathologic or diseased cell or cell type, andthe linker is cleaved by an enzyme or based on a characteristic specificfor the target cell. In some embodiments, the linker comprises an aminoacid sequence chosen from the group including AGVF, GFLG, FK, AL, ALAL,or ALALA. Other suitable linkers will be apparent to those of skill inthe art. In some embodiments, the linker is a cleavable linker. In someembodiments, the linker comprises a protease recognition site. Incertain embodiments, the linker is a UV-cleavable moiety. Suitablelinkers, for example, linkers comprising a protease recognition site, orlinkers comprising a UV cleavable moiety are known to those of skill inthe art. In some embodiments, the agent is conjugated to the ARRDC1protein or fragment thereof, or the TSG-101 protein or fragment thereof,via a sortase reaction, and the linker comprises an LPXTG motif. Methodsand reagents for conjugating agents according to some aspects of thisinvention to proteins are known to those of skill in the art.Accordingly, suitable method for conjugating and agents to be includedin an ARMM to an ARRDC1 protein or fragment thereof or a TSG101 proteinor fragment thereof will be apparent to those of skill in the art basedon this disclosure.

Methods for isolating ARMMs are also provided herein. One exemplarymethod includes collecting the culture medium, or supernatant, of a cellculture comprising microvesicle-producing cells. In some embodiments,the cell culture comprises cells obtained from a subject, for example,cells suspected to exhibit a pathological phenotype, e.g., ahyperproliferative phenotype. In some embodiments, the cell culturecomprises genetically engineered cells producing ARMMs, for example,cells expressing a recombinant ARMM protein, for example, a recombinantARRDC1 or TSG101 protein, such as an ARRDC1 or TSG101 fusion protein. Insome embodiments, the supernatant is pre-cleared of cellular debris bycentrifugation, for example, by two consecutive centrifugations ofincreasing G value (e.g., 500 G and 2000 G). In some embodiments, themethod comprises passing the supernatant through a 0.2 μm filter,eliminating all large pieces of cell debris and whole cells. In someembodiments, the supernatant is subjected to ultracentrifugation, forexample, at 120,000 g for 2 h, depending on the volume of centrifugate.The pellet obtained comprises microvesicles. In some embodiments,exosomes are depleted from the microvesicle pellet by staining and/orsorting (e.g., by FACS or MACS) using an exosome marker as describedherein. Isolated or enriched ARMMs can be suspended in culture media ora suitable buffer, as described herein.

Fusion Proteins

Some aspects of this invention provide ARRDC1 fusion proteins thatcomprise an ARRDC1 protein or a fragment thereof, and a polypeptideconjugated to the ARRDC1 protein or fragment thereof. In someembodiments, the ARRDC1 fragment comprises a PSAP motif or domain(comprising the amino acid sequence PSAP). In some embodiments, theARRDC1 protein fragment comprises the PSAP motif and at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, at least 110, at least 120,at least 130, at least 140, at least 150, at least 160, at least 170, atleast 180, at least 190, at least 200, at least 210, at least 220, atleast 230, at least 240, at least 250, at least 260, at least 270, atleast 280, at least 290, or at least 300 contiguous amino acids of theARRCD1 sequence.

Some aspects of this invention provide TSG101 fusion proteins,comprising an TSG101 protein or a fragment thereof, and a polypeptideconjugated to the TSG101 I protein or fragment thereof. In someembodiments, the TSG101 fragment comprises a UEV domain. UEV domains arewell known to those of skill in the art, and exemplary UEV domains aredescribed herein (e.g., the 145 N-terminal amino acids of the human,rat, and mouse TSG101 protein sequence provided herein. Additional UEVdomain sequences will be apparent to those of skill in the art, and theinvention is not limited in this respect. In some embodiments, theTSG101 protein fragment comprises the UEV domain and at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, at least 110, at least 120,at least 130, at least 140, at least 150, at least 160, at least 170, atleast 180, at least 190, at least 200, at least 210, at least 220, atleast 230, at least 240, at least 250, at least 260, at least 270, atleast 280, at least 290, or at least 300 contiguous amino acids of theTSG101 sequence.

In some embodiments, the polypeptide is fused to the C terminus of theARRDC1 protein or protein fragment, or to the C terminus of the TSG-101protein or protein fragment. In some embodiments, the polypeptide isfused to the N terminus of the ARRDC1 protein or protein fragment, or tothe C terminus of the TSG101 protein or protein fragment.

In some embodiments, the polypeptide fused to the ARRDC1 protein orprotein fragment or to the TSG101 protein or protein fragment comprisesa transcription factor, a transcriptional repressor, a fluorescentprotein, a kinase, a phosphatase, a protease, a chromatin modulator, aligase, or a recombinase.

In some embodiments, the polypeptide is conjugated to the ARRDC1protein, the ARRDC1 protein fragment, the TSG 101 protein, or the TSG101protein fragment via a covalent bond. In some embodiments, thepolypeptide is conjugated to the ARRDC1 protein, the ARRDC1 proteinfragment, the TSG101 protein, or the TSG101 protein fragment via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the linker comprises a protease recognition site or aUV-cleavable moiety. Some embodiments, the protease recognition site isrecognized by a protease expressed in a target cell, resulting in thepolypeptide fused to the ARRDC1 protein or fragment thereof or theTSG101 protein fragment thereof being released into the cytoplasm of thetarget cell upon uptake of the ARMM.

Cells Producing Microvesicles

Some aspects of this invention provide a microvesicle-producing cellthat comprises a recombinant expression construct encoding an ARRDC1protein, or a PSAP domain-comprising fragment thereof under the controlof a heterologous promoter. Some aspects of this invention provide amicrovesicle-producing cell that comprises a recombinant expressionconstruct encoding a TSG101 protein, or a UEV domain-comprising fragmentthereof, under the control of a heterologous promoter.

In some embodiments, the expression construct comprised in themicrovesicle producing cell further encodes a polypeptide fused to theARRDC1 protein or the TSG101 protein. In some embodiments, ARMMsproduced by such a cell comprise the encoded fusion protein. In someembodiments, the polypeptide comprises a transcription factor, atranscriptional repressor, a fluorescent protein, a kinase, aphosphatase, a protease, a ligase, or a recombinase.

In some embodiments, the cell further comprises a recombinant expressionconstruct encoding a protein. In some embodiments, the encoded proteincomprises a transcription factor, a transcriptional repressor, afluorescent protein, a chromatin modulator, a kinase, a phosphatase, aprotease, a ligase, or a recombinase. ARMMs produced by such a celltypically comprise the protein encoded by the expression construct.

In some embodiments, the cell further comprises a recombinant expressionconstruct encoding an RNAi agent. In some embodiments, ARMMs produced bysuch a cell comprise the RNAi agent encoded by the expression construct.In some embodiments, the RNAi agent is an RNAi agent as describedherein, for example, a nucleic acid, a non-coding RNA, an antisense RNA,a small RNA, an siRNA, an shRNA, a microRNA, an snRNA, a snoRNA, alincRNA, or a precursor thereof.

In some embodiments, the cell further comprises a recombinant expressionconstruct encoding a ribozyme. In some embodiments, the ribozyme is acis-splicing ribozyme. In some embodiments, the ribozyme is atrans-splicing ribozyme.

In some embodiments, the expression construct is stably inserted intothe genome of the cell. In some embodiments, the expression construct ismaintained in the cell, but not inserted into the genome of the cell. Insome embodiments, the expression construct is comprised in a vector, forexample, a plasmid vector, a cosmid vector, a viral vector, or anartificial chromosome. In some embodiments, the expression constructfurther comprises additional sequences or elements that facilitate themaintenance and/or the replication of the expression construct in themicrovesicle-producing cell, or that improve the expression of theARRDC1 protein or fragment thereof in the cell. Such additionalsequences or elements may include, for example, an origin ofreplication, and antibiotic resistance cassette, a poly a sequence, or atranscriptional isolator. Some expression constructs suitable for thegeneration of microvesicle producing cells according to aspects of thisinvention are described elsewhere herein. Methods and reagents for thegeneration of additional expression constructs suitable for thegeneration of microvesicle producing cells according to aspects of thisinvention will be apparent to those of skill in the art based on thepresent disclosure.

In some embodiments, the microvesicle producing cell is a mammaliancell, for example, a mouse cell, a rat cell, a hamster cell, a rodentcell, or a nonhuman primate cell. In some embodiments, the microvesicleproducing cell is a human cell.

Methods of Microvesicle-Mediated Agent Delivery

Some aspects of this invention provide a method of delivering an agent,for example, a therapeutic agent, a small molecule, a protein, or anucleic acid, to a target cell. The target cell can be contacted with anARMM in different ways. For example, in some embodiments, a target cellis contacted directly with an ARMM as described herein, for example,with an isolated ARMM shed by a microvesicle producing cell, or with anisolated ARMM. The contacting can be done in vitro by administering theARMM to the target cell in a culture dish, or in vivo by administeringthe ARMM to a subject harboring the target cell. Alternatively, thetarget cell can be contacted with a microvesicle producing cell asdescribed herein, for example, in vitro by co-culturing the target celland the microvesicle producing cell, or in vivo by administering amicrovesicle producing cell to a subject harboring the target cell.

Accordingly, in some embodiments, the method comprises contacting thetarget cell with a microvesicle, for example, an ARMM comprising theagent to be delivered, as described herein. In some embodiments, themethod comprises contacting the target cell with amicrovesicle-producing cell as described herein. In some embodiments,the method comprises contacting the target cell with an isolatedmicrovesicle that comprises a lipid bilayer, air ARRDC1 protein orfragment thereof, and the agent.

In some embodiments, the target cell is a mammalian cell, for example, amouse cell, a rat cell, hamster cell, a rodent cell, or a nonhumanprimate cell. In some embodiments, the target cell is a human cell. Insome embodiments, the target cell is a stem cell. In some embodiments,the target cell is a cell in vitro, and the method comprisesadministering the microvesicle to the cell in vitro, or co-culturing thetarget cell with the microvesicle-producing cell in vitro. In someembodiments, the target cell is a cell in a subject, and the methodcomprises administering the microvesicle or the microvesicle-producingcell to the subject.

In some embodiments, the subject is a mammalian subject, for example, arodent, a mouse, a rat, a hamster, or a non-human primate. In someembodiments, the subject is a human subject.

In some embodiments, the target cell is a pathological cell. In someembodiments, the target cell is a cancer cell. In some embodiments, themicrovesicle is conjugated to a binding agent that selectively binds anantigen of the target cell. In some embodiments, the antigen of thetarget cell is a cell surface antigen. In some embodiments, the bindingagent is a membrane-bound immunoglobulin, an integrin, a receptor, or areceptor ligand. Suitable surface antigens of target cells, for exampleof specific target cell types, e.g. cancer cells, are known to those ofskill in the art, as are suitable binding agents that specifically bindsuch antigens. Methods for producing membrane-bound binding agents, forexample, membrane-bound immunoglobulin, for example membrane-boundantibodies or antibody fragments specifically binding a surface antigenexpressed on the cell surface of cancer cells, are also known to thoseof skill in the art. The choice of the binding agent will depend, ofcourse, on the identity or the type of the target cell. Cell surfaceantigens specifically expressed on various types of cells that can betargeted by ARMMs comprising membrane-bound binding agents will beapparent to those of skill in the art. It will be appreciated, that thepresent invention is not limited in this respect.

Co-Culture Systems

Some aspects of this invention provide in vitro cell culture systemscomprising at least two types of cells: microvesicle producing cells andtarget cells that take up the microvesicles produced. Accordingly, inthe co-culture systems provided herein, there is a shuffling of thecontents of the microvesicles produced to the target cells. Suchco-culture systems allow for the expression of a gene product ormultiple gene products generated by the microvesicle producing cells inthe target cells without genetic manipulation of the target cells.

In some embodiments, a co-culture system is provided that comprises (a)a microvesicle-producing cell population, comprising a recombinantexpression construct encoding (i) an ARRDC1 protein or fragment thereofunder the control of a heterologous promoter, and/or (ii) a TSG101protein or fragment thereof under the control of a heterologouspromoter; and (b) a target cell population. In some embodiments, theARRDC1 fragment comprises a PSAP motif, and/or the TSG101 fragmentcomprises a UEV domain. In some embodiments, the expression constructfurther encodes a polypeptide fused to the ARRDC1 protein or fragmentthereof. In some embodiments, the expression construct further encodes apolypeptide fused to the TSG101 protein or fragment thereof. In someembodiments, the host cell comprises a plurality of expressionconstructs encoding a plurality of ARRDC1 fusion proteins and/or TSG101fusion proteins. In some embodiments, the polypeptide fused to theARRDC1 protein or fragment, or to the TSG101 protein or fragment,independently comprises a transcription factor, a transcriptionalrepressor, a fluorescent protein, a kinase, a phosphatase, a protease, achromatin modulator, a ligase, or a recombinase.

One exemplary application of a co-culture system as provided herein isthe programming or reprogramming of a target cell without geneticmanipulation. For example, in some embodiments, the target cell if adifferentiated cell, for example, a fibroblast cell. In someembodiments, the microvesicle producing cells, are feeder cells, ornon-proliferating cells. In some embodiments, the microvesicle producingcells produce ARMMs comprising a reprogramming factor, or a plurality ofreprogramming factors, either as isolated proteins or as fusion proteinswith ARRDC1 or TSG101 proteins or fragments. In some embodiments,co-culture of the differentiated target cells with the microvesicleproducing cells results in the reprogramming of the differentiatedtarget cells to an embryonic state. In some embodiments, co-culture ofthe differentiated target cells with the microvesicle producing cellsresults in the programming, or trans-differentiation, of the targetcells to a differentiated cell states that is different from theoriginal cell state of the target cells. For example, in someembodiments, the target cells are fibroblast cells, and the microvesicleproducing cells express a transcription factor the expression of whichcan reprogram fibroblast cells to a neuronal state.

Another exemplary application of a co-culture system as provided hereinis the directed differentiation of embryonic stem cells. In someembodiments, the target cells are undifferentiated embryonic sterncells, and the microvesicle producing cells express one or moredifferentiation factors, for example signaling molecules, ortranscription factors, that trigger or facilitate the differentiation ofthe embryonic stem cells into differentiated cells of a desired lineage,for example neuronal cells, or mesenchymal cells.

Yet another exemplary application of a co-culture system as providedherein is the maintenance of stem cells, for example, of embryonic stemcells or of adult stem cells in an undifferentiated state in vitro. Insome such embodiments, the microvesicle producing cells expresssignaling molecules and/or transcription factors that promote stem cellmaintenance and/or inhibit stem cell differentiation. In someembodiments, the microvesicle producing cells thus create amicroenvironment for the stem cells that mimics a naturally occurringstem cell niche in vitro.

In some embodiments, the microvesicle-producing cell is a mammaliancell. In some embodiments, the microvesicle-producing cell is anon-proliferating cell. In some embodiments, the microvesicle-producingcell is a feeder cell. In some embodiments, the target cell is amammalian cell. In some embodiments, the target cell is a human cell. Insome embodiments, the target cell is a differentiated cell. In someembodiments, the target cell is a stem cell.

Methods of Microvesicle-Based Diagnostics

Some aspects of this invention provide a method of detecting a conditionin a subject based on the presence or absence of ARMMs, or based on thepresence or absence of a biomarker or a biomarker profile in ARMMsobtained from the subject. In some embodiments, the method comprises (a)obtaining an ARMM from the subject; and (b) detecting a biomarkerprofile indicative of the condition in the ARMM obtained from thesubject; wherein the presence of the biomarker profile in the ARMMobtained from the subject indicates the presence of the condition in thesubject.

Some aspects of this invention provide a method of detecting apathological cell or cell population in a subject based on the presenceor absence or the level of ARMMs, or based on the presence or absence ofa biomarker or a biomarker profile in ARMMs obtained from the subject.In some embodiments, the method comprises (a) detecting a level of ARMMsproduced by a cell or cell population obtained from the subject; and (b)comparing the level of ARMMs of (a) to a control level, wherein, if thelevel of ARMMs produced by the cell or cell population obtained from thesubject is higher than the control level, then the cell or cellpopulation is indicated to be a pathological cell or cell population. Insome embodiments, the method comprises (a) obtaining an ARMM from thesubject; and (b) detecting a biomarker profile indicative of thepathological cell or cell population in the ARMM obtained from thesubject; wherein the presence of the biomarker profile in the ARMMobtained from the subject indicates the presence of the pathologicalcell or cell population condition in the subject.

In some embodiments, the method further comprises obtaining a samplefrom the subject that comprises an ARMM. In some embodiments, the methodfurther comprises obtaining a sample from the subject comprising a cellor cell population producing ARMMs. In some embodiments, thepathological cell or cell population is a malignant cell or cellpopulation. In some embodiments, the pathological cell or cellpopulation produces an increased amount of ARMMs as compared to a normalor control cell or cell population. In some embodiments, a subjecthaving a disease harbors an increased amount of ARMMs in the diseasedtissue, or systemically, as compared to a healthy or control subject. Insome embodiments, an increased amount of ARMMs is amount of ARMMs thatis increased at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 10-fold, at leastabout 20-fold, at least about 25-fold, at least about 50-fold, at leastabout 100-fold, at least about 1,000-fold, at least about 10,000-fold,at least about 100,000-fold, or at least to about 1,000,000-fold ascompared to a control cell population or control subject.

Expression Constructs

Some aspects of this invention provide an expression constructs thatinduce or facilitate the generation of ARMMs in cells harboring such aconstruct. In some embodiments, the expression constructs encode andARRDC1 protein or fragment thereof and/or a TSG101 protein or fragmentthereof. In some embodiments, overexpression of either or both of thesegene products in a cell in use or increase the production of ARMMs inthe cell, thus turning the cell into a microvesicle producing cell. Insome embodiments, the expression constructs allows for the cloning of apolypeptide to be fused to the ARRDC1 protein or protein fragments or tothe TSG101 protein or protein fragment. In some embodiments, such anexpression construct comprises a restriction or recombination sites thatallows in-frame cloning of a nucleotide sequence encoding thepolypeptide to be fused, either at the C terminus, or at the N terminusof the encoded ARRDC1 and/or TSG101 protein or protein fragment.

In some embodiments, the expression constructs comprises (a) anucleotide sequence encoding an ARRDC1 protein or fragment thereofoperably linked to a heterologous promoter, and (b) a restriction siteor a recombination site positioned adjacent to the ARRDC1-encodingnucleotide sequence allowing for the insertion of a nucleotide sequenceencoding an additional polypeptide in frame with the ARRDC1-encodingnucleotide sequence. Some aspects of this invention provide anexpression construct comprising (a) a nucleotide sequence encoding aTSG101 protein or fragment thereof operably linked to a heterologouspromoter, and (b) a restriction site or a recombination site positionedadjacent to the TSG101-encoding nucleotide sequence allowing for theinsertion of a nucleotide sequence encoding an additional polypeptide inframe with the TSG101-encoding nucleotide sequence.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the Examples below. Thefollowing Examples are intended to illustrate the benefits of thepresent invention and to describe particular embodiments, but are notintended to exemplify the full scope of the invention. Accordingly, itwill be understood that the Examples are not meant to limit the scope ofthe invention.

EXAMPLES Materials and Methods

ARMMs isolation and Transfer. Culture media atop nascent or transfectedcells were collected and precleared of cellular debris by twoconsecutive centrifugations (500 g and 2000 g). Media were then passedthrough a 0.2 μm filter (Sarstedt) and subjected to ultracentrifugationusing the Beckman LTA110 rotor in an Optima LTX centrifuge (Beckman), orusing the SW41 Ti rotor in a L8-M centrifuge (Beckman) at 120,000 g for2 h, depending on the volume of centrifugate. Media were then aspiratedand pellets were washed twice with ice cold phosphate buffered saline(PBS). ARMMs were then resuspended either in 1.3× Lithium dodecylsulfate (LDS; Invitrogen) supplemented with β-mercaptoethanol (β-ME) orin PBS. To calibrate ARMMs production with cell number, proteinconcentrations of the corresponding cytosolic lysates were measuredusing the Protein 660 nm assay (Pierce). For ARMMs transfer assays,transfected cells were washed thoroughly and seeded atop a 0.4 μmtranswell membrane (Costar) for 16 h before transferring transwell to aplate containing untransfected 293T cells. ARMMs transfer was allowed toproceed for 30 h before harvesting. Alternatively, purified ARMMsresuspended in PBS were added to culture media containing untransfected293T cells and incubated for 24-30 h before harvesting.

Immunogold labeling and electron microscopy. For immunogold staining ofbudding vesicles or vesicles in intercellular spaces, cells were washed,gently detached with 0.5 mM EDTA, and layered on a cushion of 8% PFA in0.1 M sodium phosphate buffer. Cell pellets were collected bycentrifugation at 3000 RPM for 3 min and pellets were replenished with4% PFA fixative for 2 h at room temperature before freezing andprocessing for visualization.

Yeast two-hybrid screen. The two-hybrid screen was done as previouslydescribed (6) using the UEV domain of TSG101 (amino acid residue 1 -145)as the bait.

Cell Culture and transfections, HEK293T, HeLa and A549 cells werecultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen)supplemented with 10% Fetal Bovine Serum (FBS; Invitrogen) and 20 mMHEPES (Invitrogen). Cells were maintained at 37° C. with 5% CO₂. Plasmidtransfections were carried out using Fugene 6 (Roche). siRNAtransfections used Dharmafect I (Dharmacon).

Plasmid constructs. HA-and Flag-tagged ARRDC1 expression constructs havebeen described previously (40). Mutant derivatives of the constructswere made by site-directed mutagenesis (QuikChange kit; Stratagene):EGFP fusion constructs were made by cloning of target genes intopEGFP-N1 (CLONTECH). mCherry-expressing constructs were made by cloningof fused mCherry-target gene fragments into pCI-neo vector (Promega).pEGFPN1-VPS4a and the VPS4a-E228Q dominant negative mutant constructswere provided by Wesly Sundquist (University of Utah). GFP-tagged WWP2constructs were obtained from Daniela Rotin (University of Toronto)

Antibodies. Antibodies were obtained as follows: anti-FLAGHRP-conjugated (Sigma); anti-HA HRP-conjugated (Roche); anti-β-actin(Santa Cruz Biotech); anti-GFP (Invitrogen); anti-TSG101 (Genetex);anti-LAMP1 (Transduction Labs); anti-LAMP3/CD63 (for immunoblotting orimmunogold labeling: Santa Cruz Biotech; for immunogold labeling: BDPharmingen); anti-mCherry (Genetex); anti-WWP2 (Bethel Labs);anti-ARRDC1 (raised by us in rabbits against purified 6xHis-ARRDC1). Forimmunoprecipitation, anti-FLAG M2 or anti-HA EZview beads (Sigma) wereused as indicated.

Western blotting and immunoprecipitation. Cells were lysed in NP-40lysis buffer (0.5% NP-40, 50 mM TrisCl, 150 mM NaCl) supplemented with aprotease inhibitor cocktail (Roche). Lysates or ARMMs resuspended in LDSsample buffer were typically resolved on a 4-12% Nupage gel (Invitrogen)using MOPS running buffer supplemented with Nupage antioxidant(Invitrogen) before transfer onto a nitrocellulose membrane (GEAmersham) and probing with the indicated antibodies. Forimmunoprecipitation of cytosolic proteins, 200-300 μg of cell lysate wasprecleared with protein A agarose beads (Santa Cruz Biotech) followed byovernight incubation at 4° C. with bait-specific antibodies conjugatedto beads. Beads are then washed twice for 10 mins in lysis buffer andeluted in 2× LDS sample buffer supplemented with 10% β-ME for Westernblot analysis. For immunoprecipitation of ARMMs associated proteins,ARMMs were prepared as described above and then lysed in NP-40 lysisbuffer for immunoprecipitation of the bait, as described for cellularproteins.

Confocal microscopy. Cells grown on glass cover slips were washed twicewith PBS then incubated in 4% paraformaldehyde (PFA) for 10 min at roomtemperature. To quench PFA-derived auto-fluorescence cells were washedthree times in Tris buffered saline (TBS) containing 1 mM CaCl₂ beforebeing mounted on glass slides using the Vectashield mounting medium(Vector Labs). Image acquisition was carried out using a Leica TCS-NTlaser scanning confocal microscope (Leica) equipped with air-cooledargon and krypton lasers. Images were then processed using ImageJ (NIH).

Electron microscopy. For microvesicle visualization, an aliquot fromARMMs preparation in PBS (5-10 μl) was adsorbed for 1 min to acarbon-coated grid. Excess liquid was removed with Whatman #1 filterpaper (Whatman) followed by staining for 30 s with 0.75% uranyl formate.Adsorbed ARMMs were examined on a JEOL 1200EX transmission electronmicroscope and images were recorded with an AMT 2k CCD camera. Forimmunogold staining of ARMMs, a microvesicle pellet was fixed in 4% PFAfor 2 h, resuspended in 20% gelatin and infiltrated with 2.3 M sucrosein PBS containing 0.2 M glycine for 15 min to quench free aldehydegroups. Frozen samples were sectioned (80-90 nm sections) at −120° C.and sections were transferred to formvar carbon-coated grids forstaining. Primary staining was carried out in 1% BSA with the indicatedantibodies before washing four times with PBS and incubation withProtein A gold beads (5 nm). Stained grids were contrasted and embeddedwith 0.3% uranyl acetate in 2% methyl cellulose for 10 rains. Grids wereexamined as described above. For visualization of ARMMs budding and forassessment of ARMMs numbers (FIG. 2F), transfected cells were incubatedin fixing solution (2.5% glutaraldehyde, 1.25% PFA, and 0.03% picricacid in 0.1M sodium cacodylate buffer, pH 7.4) for 2 h at roomtemperature. The cell pellet was then washed in 0.1 M cacodylate buffer,post-fixed in a solution containing osmium tetroxide (OsO₄) and 1.5%potassium ferrocyanide, and then treated with 1% aqueous uranyl acetate.Samples were then dehydrated, treated for 1 h with propylene oxide andsoaked overnight in a 1:1 mixture of propylene oxide and TAAB Epon(Marivac Canada) before polymerization in TAAB Epon at 60° C. for 48 h.Ultrathin sections (80-90 nm) were obtained using a Reichert Ultracut-Smicrotome and mounted on copper grids stained with lead citrate. Sampleswere examined as above.

Results

ARRDC1. recruits TSG101 to the plasma membrane. Using a yeast two-hybridscreen and the UEV domain of TSG101 as the bait, we identified twoindependent clonal isolates that correspond to the C-terminal fragmentsof human arrestin-domain-containing, protein, ARRDC1 (FIG. 1A). Thisfinding is consistent with a previous study that reportedco-immunoprecipitation of ARRDC1 with TSG-101 (28). ARRDC1 contains ahighly conserved PSAP motif (FIG. 1B), and mutational alteration of thismotif to PAAP markedly reduced the interaction between ARRDC1 and TSC101(FIG. 1C). Additionally a mutation of amino acid residue methionine 95of TSG-101, which is known to be required for PS/TAP binding (7, 8),abolished the ARRDC1/TSG101 interaction (FIG. 1D). Together, theseresults demonstrate that ARDDC1 interacts with TSG101 through specificUEV/PSAP binding.

To better understand the biological consequences of the ARRDC1/TSG101interaction, we investigated the cellular locations of individuallyexpressed or co-expressed proteins that were differentially labeled(ARRDC1-GFP and mCherry-TSG-101). We discovered that ARRDC1-GFPlocalized almost exclusively to the plasma membrane (FIG. 1E), a findingthat was reproduced using Flag-tagged ARRDC1 (FIG. 7A) and in multiplecell lines tested (FIG. 7B). Consistent with our previous study (6),adventitiously expressed TSG101 was observed in the cytoplasm inmultiple punctate foci, as is characteristic of endosome-associatedlocalization (FIG. 1E). However, when co-expressed with ARRDC1, TSG101was almost completely redistributed to the plasma membrane (FIG. 1E).Importantly, co-expression of TSG101 with the PSAP mutant of ARRDC1didnot result in redistribution of717SC1101 to the cell membrane,indicating that recruitment of TSG101 to this location is dependent onthe PSAP motif in ARRDC1,

ARRDC1 drives the release of extracellular microvesicles. The dramaticrelocalization of TSG101 from endosomes to plasma membrane by ARRDC1 isreminiscent of TSG101 recruitment by the HIV Gag protein during HIVbudding (21, 22, 29). Given the striking parallel between ARRDC1 and Gagin recruitment of TSG101 from the cytosol to the plasma membrane, wehypothesized that ARRDC1 may function like Gag to mediate the directrelease of plasma membrane-derived vesicles. To test this hypothesis, weexpressed GFP, ARRDC1-GFP, or Gag-GFP in 293T cells and, 24 h later,harvested the conditioned media, and collected extracellular vesiclesthat were released by the cultured cells. Western blot analysis showedthat Gag-GET and ARRDC1-GFP proteins were robustly present inextracellular vesicles, while the control GFP protein was not (FIG. 2A),Consistent with this finding, ARRDC1 was included among the manyproteins identified by mass spectrometry in microvesicles derived fromhuman urine (30), bladder (31) and colon cancer cells (32). Electronmicroscopic examination of the released extracellular vesicles fromARRDC1-transfected cells indicated that the vesicles are less than 100nm in diameter with an average of 45 nm (FIG. 2B).

We next determined whether native ARRDC1produced endogenously isreleased into microvesicles. As shown in FIG. 2C, extracellular vesiclescollected from I-IEK293T cells contained ARRDC1 protein, indicating thatARRDC 1 release into extracellular microvesicles is a process intrinsicto these cells. Additionally, depletion of ARRDC1 by siRNA resulted inloss of the extracellular ARRDC1 immunopositive signal (FIG. 2C),indicating that the released ARRDC1 protein is of cellular origin and isnot derived from the fetal bovine serum supplementing the culture media.To further demonstrate the presence of ARRDC1 in the microvesicles wecollected, we prepared collagen-embedded sections of extracellularvesicle pellets collected from 293T cells and stained the vesicles withARRDC1 antibody. ARRDC1 was indeed detected in the microvesicles (FIG.2D, upper panel). In contrast, these microvesicles stained negative forthe exosomal marker LAMP3/CD63 (FIG. 2D, lower panel). We refer to theseextracellular microvesicles as ARRDC1 -mediated microvesicles (ARMMs).

Using a co-culture system, we observed that ARRDC1 protein containedwithin ARMMs can be transferred between cells. ARMMs donor cellstransfected with control CUT or ARRDC1-GFP were seeded atopuntransfected recipient cells on a 0.4 μm porous membrane in a transwell(see Methods). After 30 h, cell lysates were collected from both donorand recipient cells and analyzed for ARRDC1-GFP or GFP. As shown in FIG.2E, ARRDC1-GFP but not discrete GFP was detected in the recipient cells,indicating transfer of ARRDC1, which served in these experiments as asurrogate marker for ARMMs, to the recipient cells. Furthermore, theamount of ARRDC1 transferred to recipient cells was proportional to theabundance of adventitiously expressed ARRDC1 in the donor cells,providing further evidence of the functional role of ARRDC1 in ARMMsformation and indicating ARMMs-mediated protein transfer.

Release of ARMMs requires TSG-101 and the VPS4 ATPase. TSG101 isessential for MVB formation (33) and HINT budding (19). We determinedwhether the release of ARMMs also requires TSG101 . As shown in FIG. 3A,knockdown of ISG101 expression reduced ARRDC1 in ARMMs by 3 foldcompared to control. TSG101 was also released into ARMMs, as it is inviral particles produced by Gag mediated budding (34). Moreover, whenARRDC1 was reduced, TSG101 abundance in ARMMs was reduced by 70%. Thesedata demonstrate a mutual dependency of ARRDC1 and TSG101 for inclusionin ARMMs. We next determined whether ARMMs release specifically requiresthe ARRDC1/TSG101 interaction. As shown in FIG. 3B, PSAP mutant ARRDC1exhibited much decreased ARMMs release. Moreover, TSG101 wasundetectable in culture medium fractions containing vesicles produced bycells expressing the ARRDC1 PSAP mutant (FIG. 3B). Collectively, thesefindings indicate that ARMMs release requires both. TSG101 and itsinteraction with ARRDC1. Consistent with our immunogold stainingobservations (FIG. 2D), known exosomal markers LAMP3/CD63 and LAMPI werenot detected the ARRDC1-containing vesicles by Western blot analysis(FIG. 3B), indicating the non-exosomal nature of ARMMs.

The VPS4 ATPase catalyzes the final “pinch-off” of the membranes in theformation of both MVBs and viral buds (35, 36) and is another essentialcomponent of the endosomal sorting pathway. We found that this ATPase isnecessary also for ARMMs production. As shown in FIG. 3C, whereasexpression of wild type VPS4 enhanced ARMMs release, expression of acatalytically inactive mutant of the ATPase (E228Q) almost completelyblocked such release. The inhibitotry effect is greater than TSG101knockdown and may be attributable to the more potent effect by the VPS4dominant negative overexpression on the function of the ESCRT pathway.Similarly greater effects of VPS4 in comparison with TSG101 have beenobserved for HIV Gag budding (19). Together, our data argue stronglythat the ESCRT pathway is essential for the release of ARMMs, as it isfor MVB formation and viral budding.

Arrestin domain-mediated ARRDC1 localization at the plasma membrane isrequired for ARMMS release. To further establish that ARMMs are derivedfrom the plasma membrane, we examined frozen sections ofARRDC1-expressing cells by electron microscopy. Cells were transfectedwith mCherry or ARRDC1-mCherry and treated with immunogold-labeledanti-mCherry antibody. Whereas mCherry displayed ubiquitouslocalization, ARRDC1 -mCherry was limited to the cell membrane, andstaining was specifically observed in budding vesicles emanating fromthe cell surface and also in ARMMs secreted into the extracellular space(FIG. 4A). This result supports the notion that ARMMs originate from andare formed at the cell surface.

ARRDC1 contains a N-terminal domain that is highly homologous toarrestins (37). The arrestin proteins, and particularly the β-arrestins,have been shown to associate with receptors at the cell membrane toregulate their signaling (38). We hypothesized that the arrestin domainof ARRDC1 mediates its plasma membrane localization. To find highlyconserved residues that might be important for the plasma membranelocalization of ARRDC1, we aligned amino acid residues of human arrestinparalogues with ARRDC1and with another ARRDC protein, ARRDC3, which isalso known to localize to plasma membrane (39, 40) (FIG. 4B, upperpanel). Several conserved residues (F88, G180, and. N191) in thearrestin domain of ARRDC1 were identified. We mutated these conservedresidues and determined the cellular location of the correspondingARRDC1-mCherry fusion proteins. The F88L mutation completely abolishedARRDC1 association with the plasma membrane, leaving the mutant proteinevenly distributed in the cytosol, whereas the G180A and N191D mutantsretained partial plasma membrane localization (FIG. 3B). Consistent withdata shown in FIG. 1E, the plasma membrane localization of ARRDC1protein was not perturbed by mutation of PSAP to PAAP. We then examinedthe effects of arrestin domain mutants on ARMMs release. Unlike PAAPmutant ARRDC1, which localizes to the plasma membrane but is notreleased, arrestin domain mutants of ARRDC1 proteins interfered withARMMs release in a manner that correlated with the extent of disruptionof plasma membrane-association by the mutations (FIG. 4C). The F88Lmutant completely blocked ARMMs release, whereas the other two mutantshad partial inhibitory effects (FIG. 4C). These results demonstrate thatthe arrestin domain mediates ARRDC1 localization to the plasma membraneand is required for the release of ARMMs,

Ubiquitination of ARRDC1 by HECT domain ubiquitin ligase WWP2facilitates ARMMs release. ARRDC1contains two highly conserved PPXYmotifs, which in Gag and analogous viral proteins are targets forubiquitin ligases, near its C-terminus. Although cellular localizationof PPXY mutant ARRDC1 proteins was not perturbed, expression of theseproteins markedly inhibited ARMMs release (FIG. 5A). In addition, weobserved a corresponding decrease in ARRDC1 band laddering (FIG. 5A).Such laddering is suggestive of ubiquitination, and to learn whetherARRDC1 in ARMMs is in fact ubiquitinated, we co-transfected 293T cellswith HA-tagged ubiquitin and ARRDC1-GFP, and collected ARMMs forimmunoprecipitation by anti-HA antibody. As shown in FIG. 8, totallysates of ARMMs collected from ARRDC1-expressing cells also showedincreased ubiquitination, compared with a GFP control (FIG. 8)supporting the notion that the observed laddering of ARMMs-associatedARRDC1 protein results from ubiquitination and additionally suggestingthat ubiquitination facilitates ARMMs release.

To identify possible candidates for the ubiquitin E3 ligase thatmediates ARRDC1 ubiquitination and which may have a role in ARMMsrelease, anti-ARRDC1 rabbit antibody was used to immunoprecipitateendogenous ARRDC1 and associated proteins from 293T cell lysates, andimmunoprecipitates were analyzed by mass spectrometry. Among theproteins identified in ARRDC1 immunoprecipitates was WWP2, which is amember of the NEDD4 E3 ligase family (41, 42). We confirmed theinteraction between ARRDC1 and WWP2 by co-immunoprecipitation (FIG. 5B).WWP2 contains WW domains that are known to interact with PPXY motifs(41, 42). We found that mutations in the PPXY domains in ARRDC1almostcompletely inhibited its interaction with WWP2 (FIG. 5B). Additionally,as shown in FIG. 5C, ubiquitination of ARRDC1. was enhanced by WWP2 butwas not observed in cells expressing the fragment containing only the WWdomains, indicating a dominant negative effect of the WW domainexpression on ARRDC1 ubiquitination. Interestingly, both WWP2 and the WWdomain fragment were also detected in extracellular vesicle fractions,consistent with the observed WW domain-mediated interaction betweenARRDC1 and WWP2. Furthermore, a specific role for WWP2 in ARMMs releasewas demonstrated, as siRNA-mediated WWP2 knockdown markedly reduced theamount of ARRDC1 released in ARMMs (FIG. 5D). The data obtained in thisseries of experiments indicate that WWP2, through interaction with PPXYmotifs in ARRDC1, ubiquitinates ARRDC1 and enhances ARMMs release.

Identification of Proteins in ARMMs

Purification of ARMMs. ARMMs were initially collected throughultracentrifugation of 200 ml of media supernatant of actively growingHCC1419 cells. ARMMs were then subjected to further purification bysucrose gradient separation as follows. Briefly, ARMMs were re-suspendedin 0.5 ml PBS and laid on top of a sucrose step-wise gradient (from 0.2to 2 M, 1 ml of each concentration, FIG. 10). The sample was thencentrifuged in a swinging bucket (SW50.1 rotor, Beckman) at 130,000×gfor 18 hours. After centrifugation, about ten (10) fractions of 1 mlvolume were collected. A small volume (˜10 μl) of each fraction wassubjected to SDS-PAGE followed by Western blotting analysis for ARRDC1.As shown in FIG. 10, ARRDC1 was detected in mostly three sucrosefractions 1.0 and 1.2 M).

Identification of Proteins in ARMMs. Following sucrose gradientseparation, two fractions (0.8 and 1.0 M sucrose) that contain most ofARMMs (marked by arrows in FIG. 10) were combined together, diluted withPBS to a volume of 10 ml and re-centrifuged (100,000×g for 2 hours). Theresulting pellet was lysed in SDS-PAGE loading buffer, and proteins wereresolved by SDS-PAGE (12% gel) followed by coomassie-blue staining (FIG.11). About 18 distinct protein bands were identified and analyzed bymass spectrometry. Over 500 proteins were identified with at least twopeptides. ARRDC1 was identified in several fractions, consistent withresults of ARRDC1 modification by ubiquitination described herein (seealso Nabhan et al,. Formation and release of arrestin domain-containingprotein 1-mediated microvesicles (ARMMs) at plasma membrane byrecruitment of ISG101 protein. Proc Natl Acad Sci U S A. 2012;109(11):4146-51; the entire contents of which are incorporated herein byreference). The notion of ARRDC1 modification by ubiquitination isfurther supported by the identification of several ubiquitin E3 ligases(WWP1, WWP2 and ITCH) in the ARMMs (Table 1). Gene/Protein Nomenclaturefollows official guidelines for gene nomenclature (see Hester et at.,Guidelines for Human Gene Nomenclature Genomics 79(4):464-470 (2002),and the HUGO Gene Nomenclature Committee gene name database, accessibleat genenames.org).

TABLE 1 Proteins identified in ARMMs. Band # of Peptides identified inMass-Spec Gene/Protein Symbol #1 52 MYO10 22 TLN2 18 MYO18A 16 MTOR 5KIF13B 5 KIAA1244 #2 14 LNPEP 14 ERBB2IP 10 TNS3 7 MON2 9 KIAA1522 #3 36CASK 22 NOTCH2 16 AP1B1 15 USP5 9 EXOC2 7 VAV2 7 EXOC1 5 MTSS1 5 ITCH 6XPOT 5 ARRDC1 #4 21 MARK2 17 PIK3R2 16 ENAH 8 QARS 7 CNNM4 6 EFCAB4B 5KIAA1598 #5 26 EXOC7 17 ZDHHC5 14 EXOC8 13 FERMT2 12 MLPH 10 RHPN2 9 LSS9 IPI:IPI00719051.3 9 PVRL2 11 ZYX 8 ARRDC1 5 EIF4B 6 TRIM3 #6 7 SYTL1 5PVRL2 8 PKM2 6 NF2 5 GALNT7 #7 8 PTPRD 5 ARRDC1 #8 42 PKM2 22 SPAG1 18YARS 16 SNTB2 13 GRB7 9 FAM83F 10 CPM 12 ARRDC1 8 RAD23B 8 EPB41L5 6ILK-2 10 GRB7 7 SPINT1 6 FNBP1L 5 LRRC1 6 AHCYL2 #9 16 AP1M2 14 PSMC3 12IPI:IPI00328587.4 14 CXADR 19 ARRDC1 10 PARVA 8 C8orf30A 9 KIAA0174 6TUBB2C 6 OCLN # 10  10 CSNK2A1 8 INPP5A 8 MAPK3 7 PPID 7 FAM102A 7ARRDC1 8 SH3GL1 5 TUBB2C 5 KIAA0174 #11  14 STX16 8 AIP 10 FAM84B 8CSNK1A1 6 PRKAG1 #12  14 STARD10 15 LASP1 12 AKR7L 9 STX16 8 SRM 7KIAA0174 8 TWF1 6 CSNK1A1L 7 CSNK1A1 7 PPCS 5 STUB1 #13  10 VAV3

Many of the identified proteins, including CD9, TSG101, PDCD6IP,Annexins, and heat shock protein HSPA8, have previously been identifiedin exosomes. This could be due to the inclusion of some exosomes in thepurified ARMMs fractions or could suggest that ARMMs and exosomes mayshare some common components. To identify unique ARMMs proteins, theprotein list obtained from mass spec was compared with known exosomalproteins in the ExoCarta database (exocarta.org). This analysis resultedin the identification of over 100 proteins that were not found inexosomes and thus are likely unique components of ARMMs (Table 1). Amongthe proteins are several member of the exocyst complex (EXOC7, EXOC8,EXOC1, and EXOC2), which is involved in exocytosis and mediates thetethering and spatial targeting of post-Golgi vesicles to plasmamembrane. The presence of exocyst proteins in ARMMs suggests newfunction of these proteins and may point to specific targeting mechanismfor ARMMs.

ARMMs contain RNA species. HEK293 cells were first stably transducedwith ARRDC1 -shRNA to knock down endogenous ARRDC 1. ARRDC1 -knockdowncells were then transfected with nucleic acid constructs expressingeither mCherry or ARRDC1-mcherry. Supernatant medium from both types ofcells were subjected to ultracentrifugation. RNAs in centrifuged pelletswere extracted by Trizol reagent (Invitrogen) and analyzed byBioanalyzer (Agilent). While the cellular RNA profiles from mCherry- andARRDC1-mCherry-expressing cells are very similar (figure inset),supernatants from cells expressing ARRDC1-mcherry, but not the controlmCherry cells, contained detectable RNA species, including small onesranging from 50-80 nucleotides. Delivery of these RNA species throughARMMs to target cell or tissue may mediate specific biologicalfunctions.

Discussion

Mammalian cells are capable of secreting into the extracellular milieu avariety of microvesicles, among which are particles termed exosomes,which are derived from MVBs of late endosomes (2, 14, 43). The resultswe report reveal a type of microvesicle that is generated by directplasma membrane budding (DPMB) and is distinct from exosomes. WhereasTSG101 is located at the surface of late endosomes during the formationof MVBs, TSG-101 is recruited by ARRDC1 to the surface of cells forproduction of ARMMs. Consistent with the plasma membrane origin ofARMMs, our data show that ARMMs lack late endosomal markers such as CD63and LAMP1 (FIG. 2D and FIG. 3B).

Our findings suggest a direct analogy between ARMMs formation andbudding mediated by Gag and other viral proteins (FIG. 6). It has beenwell established that many viruses including HIV utilize cellularmachinery for their egress (16-18), HIV Gag uses PSAP motifs to recruitTSG101 and other endosomal pathway proteins to bud from the cellsurface. It has been suggested that Gag protein functionally mimics hostprotein Hrs, which interacts with TSG-101 through a PSAP motif (11).However, while Hrs and Gag interact with TSG101 Hrs is an earlyendosome-associated protein and does not localize to the plasmamembrane, where viral budding occurs (6, 44). In contrast, ARRDC1 isdirected to the plasma membrane by its arrestin-like domain andmultimerizes there (FIG. 9) , as does Gag (45), Moreover, both Gag andARRDC1, when adventitiously expressed, are sufficient to induce vesicleformation. Additionally, our results demonstrate that like HIV Gagbudding, ARMMs release requires both TSG101 and VPS4. These datastrongly suggest that HIV Gag and likely other viral proteins mimicARRDC1 to mediate the release of viral particles from host cells.Whether ARMMs production also requires ESCRT-III and Alix, which areinvolved in Gag-mediated HIV budding (46), currently is not known.Recently, the shedding of ESCRT-dependent microvesicles that may proveto be ARMMs has been found to be increased in elegans embryos by loss ofthe evolutionarily-conserved P4-ATPase, TAT-5, and consequent exposureof phosphaidylethanolamine at the cell surface (47),

ARRDC1belongs to a family of ARRDC proteins that share a common domainstructure: an arrestin domain at the N-termini and two PPXY motifs atthe C-termini (37). ARRDC1contains a highly conserved PSAP motif that isnot found in other ARRDC proteins. The PSAP motif directly interactswith TSG101, and in cells that adventitiously overexpress ARRDC1,redirects TSG101 from endosomes to the cell surface. Potentially, suchARRDC1-mediated relocalization of TSG101 may alter endosomal traffickingand sorting and consequently, signal transduction by receptors subjectedto endosomal sorting mechanisms. As the arrestin domain of ARRDC1mediates localization of this protein to the plasma membrane, and asβ-arrestins are known to bind to phosphorylated receptor proteins (38),ARRDC1 may prove to be a multifaceted regulator of receptor-mediatedsignaling.

Our data also indicate a role for ARRDC1 ubiquitination in ARMMsrelease. We showed that ARRDC1 PPXY motifs interact with the ubiquitinE3 ligase WWP2 (FIG. 5B), and that WWP2 mediates the ubiquitination ofARRDC1—in particular the ARDDC1 species that are incorporated intoARMMs. A recent study has shown that another E3 ligase WWP1 can alsointeract with and ubiquitinate ARRDC1 (28). It remains unclear whetherWWP1 also has a role in ARMMs production. Interestingly, our previousstudy has shown that the ARRDC1 relative, ARRDC3, also interacts withmembers of the NI E3 ligase family (40). Through interaction with NEDD4E3 ligases, ARRDC3 mediates ubiquitination of β2-adrenergic receptors.

Extracellular microvesicles have the potential to function as mediatorsof cell-cell communication (2). Indeed, several studies have identifiedfunctional proteins and RNA molecules from exosomal microvesicles andhave shown that macromolecules transferred by exosomes can producefunctional effects in recipient cells (48, 49). ARMMs may functionsimilarly in cellular communication, and consistent with this notion, wefound that ARMMs can transfer ARRDC1 between cells. ARMMs, likeexosomes, may contain RNAs or additional proteins involved in cell-cellcommunication, and given the ability of ARRDC1 to localize to the plasmamembrane, some of these proteins may be plasma membrane receptors.

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The contents of all publications, patents, websites, and databaseentries mentioned herein, including references 1-49 listed above, arehereby incorporated by reference in their entirety as if each individualpublication, patent, website, and database entry was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process.

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the claims or from relevant portions of the description isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claim that is dependent on the same base claim.Furthermore, where the claims recite a composition, it is to beunderstood that methods of using the composition for any of the purposesdisclosed herein are included, and methods of making the compositionaccording to any of the methods of making disclosed herein or othermethods known in the art are included, unless otherwise indicated orunless it would be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodimentdescribed herein may be explicitly excluded from any one or more of theclaims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the invention, can be excluded from any one or moreclaims. For purposes of brevity, all of the embodiments in which one ormore elements, features, purposes, or aspects is excluded are not setforth explicitly herein.

1-29. (canceled)
 30. An ARRDC1 fusion protein, comprising an ARRDC1protein or a fragment thereof, and a polypeptide conjugated to theARRDC1 protein or fragment thereof.
 31. The ARRDC1 fusion protein ofclaim 30, wherein the ARRDC1 fragment comprises a PSAP domain.
 32. ATSG101 fusion protein, comprising an TSG101 protein or a fragmentthereof, and a polypeptide conjugated to the TSG101 protein or fragmentthereof.
 33. The TSG101 fusion protein of claim 32, wherein the TSG101fragment comprises a UEV domain.
 34. The fusion protein of claim 30,wherein the polypeptide comprises a transcription factor, atranscriptional repressor, a fluorescent protein, a kinase, aphosphatase, a protease, a ligase, or a recombinase.
 35. The fusionprotein of claim 30, wherein the polypeptide is conjugated to the ARRDC1protein or the ARRDC1 protein fragment via a covalent bond.
 36. Thefusion protein of claim 30, wherein the polypeptide is conjugated to theARRDC1 protein or the ARRDC1 protein fragment via a linker.
 37. Thefusion protein of claim 36 wherein the linker is a cleavable linker. 38.The fusion protein of claim 37, wherein the linker comprises a proteaserecognition site or a UV-cleavable moiety.
 39. The fusion protein ofclaim 30, wherein the polypeptide comprises a fluorescent protein.40-86. (canceled)
 87. The fusion protein of claim 32, wherein thepolypeptide comprises a transcription factor, a transcriptionalrepressor, a fluorescent protein, a kinase, a phosphatase, a protease, aligase, or a recombinase.
 88. The fusion protein of claim 32, whereinthe polypeptide is conjugated to the TSG101 protein or the TSG101protein fragment via a covalent bond.
 89. The fusion protein of claim32, wherein the polypeptide is conjugated to the TSG101 protein or theTSG101 protein fragment via a linker.
 90. The fusion protein of claim 89wherein the linker is a cleavable linker.
 91. The fusion protein ofclaim 90, wherein the linker comprises a protease recognition site or aUV-cleavable moiety.
 92. The fusion protein of claim 32, wherein thepolypeptide comprises a fluorescent protein.
 93. The fusion protein ofclaim 92, wherein the fluorescent protein is GFP, YFP, BFP, RFP,mCherry, or an enhanced or destabilized version thereof.
 94. Amicrovesicle-producing cell comprising a the fusion protein of claim 30.95. A microvesicle-producing cell comprising a recombinant expressionconstruct encoding the fusion protein of claim 30 under the control of aheterologous promoter.
 96. A method of delivering an agent to a targetcell, the method comprising contacting the target cell with an isolatedmicrovesicle comprising a lipid bilayer, the fusion protein of claim 30,and the agent.